diff --git a/crates/uv-cli/src/lib.rs b/crates/uv-cli/src/lib.rs
index b57863797..c624c32dc 100644
--- a/crates/uv-cli/src/lib.rs
+++ b/crates/uv-cli/src/lib.rs
@@ -3192,8 +3192,8 @@ pub struct RunArgs {
     ///
     /// Can be provided multiple times, with subsequent files overriding values defined in previous
     /// files.
-    #[arg(long, value_delimiter = ' ', env = EnvVars::UV_ENV_FILE)]
-    pub env_file: Vec<PathBuf>,
+    #[arg(long, env = EnvVars::UV_ENV_FILE)]
+    pub env_file: Vec<String>,
 
     /// Avoid reading environment variables from a `.env` file.
     #[arg(long, value_parser = clap::builder::BoolishValueParser::new(), env = EnvVars::UV_NO_ENV_FILE)]
diff --git a/crates/uv-configuration/src/env_file.rs b/crates/uv-configuration/src/env_file.rs
new file mode 100644
index 000000000..8a7bf855d
--- /dev/null
+++ b/crates/uv-configuration/src/env_file.rs
@@ -0,0 +1,135 @@
+use std::path::PathBuf;
+
+/// A collection of `.env` file paths.
+#[derive(Default, Debug, Clone, PartialEq, Eq)]
+pub struct EnvFile(Vec<PathBuf>);
+
+impl EnvFile {
+    /// Parse the env file paths from command-line arguments.
+    pub fn from_args(env_file: Vec<String>, no_env_file: bool) -> Self {
+        if no_env_file {
+            return Self::default();
+        }
+
+        if env_file.is_empty() {
+            return Self::default();
+        }
+
+        let mut paths = Vec::new();
+
+        // Split on spaces, but respect backslashes.
+        for env_file in env_file {
+            let mut current = String::new();
+            let mut escape = false;
+            for c in env_file.chars() {
+                if escape {
+                    current.push(c);
+                    escape = false;
+                } else if c == '\\' {
+                    escape = true;
+                } else if c.is_whitespace() {
+                    if !current.is_empty() {
+                        paths.push(PathBuf::from(current));
+                        current = String::new();
+                    }
+                } else {
+                    current.push(c);
+                }
+            }
+            if !current.is_empty() {
+                paths.push(PathBuf::from(current));
+            }
+        }
+
+        Self(paths)
+    }
+
+    /// Iterate over the paths in the env file.
+    pub fn iter(&self) -> impl DoubleEndedIterator<Item = &PathBuf> {
+        self.0.iter()
+    }
+}
+
+#[cfg(test)]
+mod tests {
+    use super::*;
+
+    #[test]
+    fn test_from_args_default() {
+        let env_file = EnvFile::from_args(vec![], false);
+        assert_eq!(env_file, EnvFile::default());
+    }
+
+    #[test]
+    fn test_from_args_no_env_file() {
+        let env_file = EnvFile::from_args(vec!["path1 path2".to_string()], true);
+        assert_eq!(env_file, EnvFile::default());
+    }
+
+    #[test]
+    fn test_from_args_empty_string() {
+        let env_file = EnvFile::from_args(vec![String::new()], false);
+        assert_eq!(env_file, EnvFile::default());
+    }
+
+    #[test]
+    fn test_from_args_whitespace_only() {
+        let env_file = EnvFile::from_args(vec!["   ".to_string()], false);
+        assert_eq!(env_file, EnvFile::default());
+    }
+
+    #[test]
+    fn test_from_args_single_path() {
+        let env_file = EnvFile::from_args(vec!["path1".to_string()], false);
+        assert_eq!(env_file.0, vec![PathBuf::from("path1")]);
+    }
+
+    #[test]
+    fn test_from_args_multiple_paths() {
+        let env_file = EnvFile::from_args(vec!["path1 path2 path3".to_string()], false);
+        assert_eq!(
+            env_file.0,
+            vec![
+                PathBuf::from("path1"),
+                PathBuf::from("path2"),
+                PathBuf::from("path3")
+            ]
+        );
+    }
+
+    #[test]
+    fn test_from_args_escaped_spaces() {
+        let env_file = EnvFile::from_args(vec![r"path\ with\ spaces".to_string()], false);
+        assert_eq!(env_file.0, vec![PathBuf::from("path with spaces")]);
+    }
+
+    #[test]
+    fn test_from_args_mixed_escaped_and_normal() {
+        let env_file =
+            EnvFile::from_args(vec![r"path1 path\ with\ spaces path2".to_string()], false);
+        assert_eq!(
+            env_file.0,
+            vec![
+                PathBuf::from("path1"),
+                PathBuf::from("path with spaces"),
+                PathBuf::from("path2")
+            ]
+        );
+    }
+
+    #[test]
+    fn test_from_args_escaped_backslash() {
+        let env_file = EnvFile::from_args(vec![r"path\\with\\backslashes".to_string()], false);
+        assert_eq!(env_file.0, vec![PathBuf::from(r"path\with\backslashes")]);
+    }
+
+    #[test]
+    fn test_iter() {
+        let env_file = EnvFile(vec![PathBuf::from("path1"), PathBuf::from("path2")]);
+        let paths: Vec<_> = env_file.iter().collect();
+        assert_eq!(
+            paths,
+            vec![&PathBuf::from("path1"), &PathBuf::from("path2")]
+        );
+    }
+}
diff --git a/crates/uv-configuration/src/lib.rs b/crates/uv-configuration/src/lib.rs
index ec5514bf0..500a7aa38 100644
--- a/crates/uv-configuration/src/lib.rs
+++ b/crates/uv-configuration/src/lib.rs
@@ -5,6 +5,7 @@ pub use constraints::*;
 pub use dependency_groups::*;
 pub use dry_run::*;
 pub use editable::*;
+pub use env_file::*;
 pub use export_format::*;
 pub use extras::*;
 pub use hash::*;
@@ -28,6 +29,7 @@ mod constraints;
 mod dependency_groups;
 mod dry_run;
 mod editable;
+mod env_file;
 mod export_format;
 mod extras;
 mod hash;
diff --git a/crates/uv/src/commands/project/run.rs b/crates/uv/src/commands/project/run.rs
index e6cf2f50f..3101cee81 100644
--- a/crates/uv/src/commands/project/run.rs
+++ b/crates/uv/src/commands/project/run.rs
@@ -18,7 +18,7 @@ use uv_cache::Cache;
 use uv_cli::ExternalCommand;
 use uv_client::BaseClientBuilder;
 use uv_configuration::{
-    Concurrency, Constraints, DependencyGroups, DryRun, EditableMode, ExtrasSpecification,
+    Concurrency, Constraints, DependencyGroups, DryRun, EditableMode, EnvFile, ExtrasSpecification,
     InstallOptions, TargetTriple,
 };
 use uv_distribution::LoweredExtraBuildDependencies;
@@ -106,8 +106,7 @@ pub(crate) async fn run(
     concurrency: Concurrency,
     cache: &Cache,
     printer: Printer,
-    env_file: Vec<PathBuf>,
-    no_env_file: bool,
+    env_file: EnvFile,
     preview: Preview,
     max_recursion_depth: u32,
 ) -> anyhow::Result<ExitStatus> {
@@ -164,39 +163,37 @@ hint: If you are running a script with `{}` in the shebang, you may need to incl
     let workspace_cache = WorkspaceCache::default();
 
     // Read from the `.env` file, if necessary.
-    if !no_env_file {
-        for env_file_path in env_file.iter().rev().map(PathBuf::as_path) {
-            match dotenvy::from_path(env_file_path) {
-                Err(dotenvy::Error::Io(err)) if err.kind() == std::io::ErrorKind::NotFound => {
-                    bail!(
-                        "No environment file found at: `{}`",
-                        env_file_path.simplified_display()
-                    );
-                }
-                Err(dotenvy::Error::Io(err)) => {
-                    bail!(
-                        "Failed to read environment file `{}`: {err}",
-                        env_file_path.simplified_display()
-                    );
-                }
-                Err(dotenvy::Error::LineParse(content, position)) => {
-                    warn_user!(
-                        "Failed to parse environment file `{}` at position {position}: {content}",
-                        env_file_path.simplified_display(),
-                    );
-                }
-                Err(err) => {
-                    warn_user!(
-                        "Failed to parse environment file `{}`: {err}",
-                        env_file_path.simplified_display(),
-                    );
-                }
-                Ok(()) => {
-                    debug!(
-                        "Read environment file at: `{}`",
-                        env_file_path.simplified_display()
-                    );
-                }
+    for env_file_path in env_file.iter().rev().map(PathBuf::as_path) {
+        match dotenvy::from_path(env_file_path) {
+            Err(dotenvy::Error::Io(err)) if err.kind() == std::io::ErrorKind::NotFound => {
+                bail!(
+                    "No environment file found at: `{}`",
+                    env_file_path.simplified_display()
+                );
+            }
+            Err(dotenvy::Error::Io(err)) => {
+                bail!(
+                    "Failed to read environment file `{}`: {err}",
+                    env_file_path.simplified_display()
+                );
+            }
+            Err(dotenvy::Error::LineParse(content, position)) => {
+                warn_user!(
+                    "Failed to parse environment file `{}` at position {position}: {content}",
+                    env_file_path.simplified_display(),
+                );
+            }
+            Err(err) => {
+                warn_user!(
+                    "Failed to parse environment file `{}`: {err}",
+                    env_file_path.simplified_display(),
+                );
+            }
+            Ok(()) => {
+                debug!(
+                    "Read environment file at: `{}`",
+                    env_file_path.simplified_display()
+                );
             }
         }
     }
diff --git a/crates/uv/src/lib.rs b/crates/uv/src/lib.rs
index 95d636732..345302946 100644
--- a/crates/uv/src/lib.rs
+++ b/crates/uv/src/lib.rs
@@ -1865,7 +1865,6 @@ async fn run_project(
                 &cache,
                 printer,
                 args.env_file,
-                args.no_env_file,
                 globals.preview,
                 args.max_recursion_depth,
             ))
diff --git a/crates/uv/src/settings.rs b/crates/uv/src/settings.rs
index e3c71649e..9c1ab8f07 100644
--- a/crates/uv/src/settings.rs
+++ b/crates/uv/src/settings.rs
@@ -23,7 +23,7 @@ use uv_cli::{
 };
 use uv_client::Connectivity;
 use uv_configuration::{
-    BuildIsolation, BuildOptions, Concurrency, DependencyGroups, DryRun, EditableMode,
+    BuildIsolation, BuildOptions, Concurrency, DependencyGroups, DryRun, EditableMode, EnvFile,
     ExportFormat, ExtrasSpecification, HashCheckingMode, IndexStrategy, InstallOptions,
     KeyringProviderType, NoBinary, NoBuild, ProjectBuildBackend, Reinstall, RequiredVersion,
     SourceStrategy, TargetTriple, TrustedHost, TrustedPublishing, Upgrade, VersionControlSystem,
@@ -343,8 +343,7 @@ pub(crate) struct RunSettings {
     pub(crate) install_mirrors: PythonInstallMirrors,
     pub(crate) refresh: Refresh,
     pub(crate) settings: ResolverInstallerSettings,
-    pub(crate) env_file: Vec<PathBuf>,
-    pub(crate) no_env_file: bool,
+    pub(crate) env_file: EnvFile,
     pub(crate) max_recursion_depth: u32,
 }
 
@@ -461,8 +460,7 @@ impl RunSettings {
                 resolver_installer_options(installer, build),
                 filesystem,
             ),
-            env_file,
-            no_env_file,
+            env_file: EnvFile::from_args(env_file, no_env_file),
             install_mirrors,
             max_recursion_depth: max_recursion_depth.unwrap_or(Self::DEFAULT_MAX_RECURSION_DEPTH),
         }