diff --git a/packages/vite/src/node/plugins/optimizedDeps.ts b/packages/vite/src/node/plugins/optimizedDeps.ts
index cdde20891b836b..6d6a8d22eb9468 100644
--- a/packages/vite/src/node/plugins/optimizedDeps.ts
+++ b/packages/vite/src/node/plugins/optimizedDeps.ts
@@ -10,6 +10,8 @@ import { cleanUrl } from '../../shared/utils'
 export const ERR_OPTIMIZE_DEPS_PROCESSING_ERROR =
   'ERR_OPTIMIZE_DEPS_PROCESSING_ERROR'
 export const ERR_OUTDATED_OPTIMIZED_DEP = 'ERR_OUTDATED_OPTIMIZED_DEP'
+export const ERR_FILE_NOT_FOUND_IN_OPTIMIZED_DEP_DIR =
+  'ERR_FILE_NOT_FOUND_IN_OPTIMIZED_DEP_DIR'
 
 const debug = createDebugger('vite:optimize-deps')
 
@@ -68,8 +70,12 @@ export function optimizedDepsPlugin(config: ResolvedConfig): Plugin {
         try {
           return await fsp.readFile(file, 'utf-8')
         } catch (e) {
-          // Outdated non-entry points (CHUNK), loaded after a rerun
-          throwOutdatedRequest(id)
+          const newMetadata = depsOptimizer.metadata
+          if (optimizedDepInfoFromFile(newMetadata, file)) {
+            // Outdated non-entry points (CHUNK), loaded after a rerun
+            throwOutdatedRequest(id)
+          }
+          throwFileNotFoundInOptimizedDep(id)
         }
       }
     },
@@ -97,3 +103,15 @@ export function throwOutdatedRequest(id: string): never {
   // send a 504 status code request timeout
   throw err
 }
+
+export function throwFileNotFoundInOptimizedDep(id: string): never {
+  const err: any = new Error(
+    `The file does not exist at "${id}" which is in the optimize deps directory. ` +
+      `The dependency might be incompatible with the dep optimizer. ` +
+      `Try adding it to \`optimizeDeps.exclude\`.`,
+  )
+  err.code = ERR_FILE_NOT_FOUND_IN_OPTIMIZED_DEP_DIR
+  // This error will be caught by the transform middleware that will
+  // send a 404 status code not found
+  throw err
+}
diff --git a/packages/vite/src/node/server/middlewares/transform.ts b/packages/vite/src/node/server/middlewares/transform.ts
index a0239aab7fcb4e..12a440d4c10774 100644
--- a/packages/vite/src/node/server/middlewares/transform.ts
+++ b/packages/vite/src/node/server/middlewares/transform.ts
@@ -27,6 +27,7 @@ import {
   isDirectRequest,
 } from '../../plugins/css'
 import {
+  ERR_FILE_NOT_FOUND_IN_OPTIMIZED_DEP_DIR,
   ERR_OPTIMIZE_DEPS_PROCESSING_ERROR,
   ERR_OUTDATED_OPTIMIZED_DEP,
 } from '../../plugins/optimizedDeps'
@@ -253,6 +254,15 @@ export function transformMiddleware(
         // error but a normal part of the missing deps discovery flow
         return
       }
+      if (e?.code === ERR_FILE_NOT_FOUND_IN_OPTIMIZED_DEP_DIR) {
+        // Skip if response has already been sent
+        if (!res.writableEnded) {
+          res.statusCode = 404
+          res.end()
+        }
+        server.config.logger.warn(colors.yellow(e.message))
+        return
+      }
       if (e?.code === ERR_LOAD_URL) {
         // Let other middleware handle if we can't load the url via transformRequest
         return next()
diff --git a/playground/optimize-deps/__tests__/optimize-deps.spec.ts b/playground/optimize-deps/__tests__/optimize-deps.spec.ts
index f2ad3680cdac47..b07e5f8fe59a2a 100644
--- a/playground/optimize-deps/__tests__/optimize-deps.spec.ts
+++ b/playground/optimize-deps/__tests__/optimize-deps.spec.ts
@@ -319,3 +319,11 @@ test('long file name should work', async () => {
     `hello world`,
   )
 })
+
+test.runIf(isServe)('warn on incompatible dependency', () => {
+  expect(serverLogs).toContainEqual(
+    expect.stringContaining(
+      'The dependency might be incompatible with the dep optimizer.',
+    ),
+  )
+})
diff --git a/playground/optimize-deps/dep-incompatible/index.js b/playground/optimize-deps/dep-incompatible/index.js
new file mode 100644
index 00000000000000..6d67368a1d4df7
--- /dev/null
+++ b/playground/optimize-deps/dep-incompatible/index.js
@@ -0,0 +1,3 @@
+const subUrl = new URL('./sub.js', import.meta.url)
+
+export default () => import(subUrl)
diff --git a/playground/optimize-deps/dep-incompatible/package.json b/playground/optimize-deps/dep-incompatible/package.json
new file mode 100644
index 00000000000000..1d67c51eb4cbcc
--- /dev/null
+++ b/playground/optimize-deps/dep-incompatible/package.json
@@ -0,0 +1,7 @@
+{
+  "name": "@vitejs/test-dep-incompatible",
+  "private": true,
+  "version": "0.0.0",
+  "type": "module",
+  "main": "index.js"
+}
diff --git a/playground/optimize-deps/dep-incompatible/sub.js b/playground/optimize-deps/dep-incompatible/sub.js
new file mode 100644
index 00000000000000..26f9a8a9430bf8
--- /dev/null
+++ b/playground/optimize-deps/dep-incompatible/sub.js
@@ -0,0 +1 @@
+export default 'sub'
diff --git a/playground/optimize-deps/index.html b/playground/optimize-deps/index.html
index 3839a4aa9951d0..9b992b7dd4892a 100644
--- a/playground/optimize-deps/index.html
+++ b/playground/optimize-deps/index.html
@@ -263,3 +263,8 @@ <h2>Long file name import works</h2>
   text('.clonedeep-slash', cloneDeepSlash({ name: 'clonedeep-slash' }).name)
   text('.clonedeep-dot', cloneDeepDot({ name: 'clonedeep-dot' }).name)
 </script>
+
+<script type="module">
+  import loadSub from '@vitejs/test-dep-incompatible'
+  loadSub() // should show an error that tells there's an incompatible dep
+</script>
diff --git a/playground/optimize-deps/package.json b/playground/optimize-deps/package.json
index a1cf32ebc73a22..99be592eb4972f 100644
--- a/playground/optimize-deps/package.json
+++ b/playground/optimize-deps/package.json
@@ -20,6 +20,7 @@
     "@vitejs/test-dep-cjs-with-assets": "file:./dep-cjs-with-assets",
     "@vitejs/test-dep-css-require": "file:./dep-css-require",
     "@vitejs/test-dep-esbuild-plugin-transform": "file:./dep-esbuild-plugin-transform",
+    "@vitejs/test-dep-incompatible": "file:./dep-incompatible",
     "@vitejs/test-dep-linked": "link:./dep-linked",
     "@vitejs/test-dep-linked-include": "link:./dep-linked-include",
     "@vitejs/test-dep-node-env": "file:./dep-node-env",
diff --git a/pnpm-lock.yaml b/pnpm-lock.yaml
index 03b925b3a34c11..929c39d3b8ce48 100644
--- a/pnpm-lock.yaml
+++ b/pnpm-lock.yaml
@@ -906,6 +906,9 @@ importers:
       '@vitejs/test-dep-esbuild-plugin-transform':
         specifier: file:./dep-esbuild-plugin-transform
         version: file:playground/optimize-deps/dep-esbuild-plugin-transform
+      '@vitejs/test-dep-incompatible':
+        specifier: file:./dep-incompatible
+        version: file:playground/optimize-deps/dep-incompatible
       '@vitejs/test-dep-linked':
         specifier: link:./dep-linked
         version: link:dep-linked
@@ -1019,6 +1022,8 @@ importers:
 
   playground/optimize-deps/dep-esbuild-plugin-transform: {}
 
+  playground/optimize-deps/dep-incompatible: {}
+
   playground/optimize-deps/dep-linked:
     dependencies:
       lodash-es:
@@ -9901,6 +9906,11 @@ packages:
     name: '@vitejs/test-dep-esbuild-plugin-transform'
     dev: false
 
+  file:playground/optimize-deps/dep-incompatible:
+    resolution: {directory: playground/optimize-deps/dep-incompatible, type: directory}
+    name: '@vitejs/test-dep-incompatible'
+    dev: false
+
   file:playground/optimize-deps/dep-node-env:
     resolution: {directory: playground/optimize-deps/dep-node-env, type: directory}
     name: '@vitejs/test-dep-node-env'