diff --git a/.changeset/wet-chefs-love.md b/.changeset/wet-chefs-love.md
new file mode 100644
index 000000000000..4ffc4a7657f7
--- /dev/null
+++ b/.changeset/wet-chefs-love.md
@@ -0,0 +1,5 @@
+---
+'astro': patch
+---
+
+Fix `astro:server:setup` middlewares not applying. This resolves an issue with the Partytown integration in dev.
diff --git a/packages/astro/src/core/create-vite.ts b/packages/astro/src/core/create-vite.ts
index e658a0c76be3..ab7208b7d01c 100644
--- a/packages/astro/src/core/create-vite.ts
+++ b/packages/astro/src/core/create-vite.ts
@@ -19,6 +19,7 @@ import envVitePlugin from '../vite-plugin-env/index.js';
 import astroHeadPlugin from '../vite-plugin-head/index.js';
 import htmlVitePlugin from '../vite-plugin-html/index.js';
 import { astroInjectEnvTsPlugin } from '../vite-plugin-inject-env-ts/index.js';
+import astroIntegrationsContainerPlugin from '../vite-plugin-integrations-container/index.js';
 import jsxVitePlugin from '../vite-plugin-jsx/index.js';
 import astroLoadFallbackPlugin from '../vite-plugin-load-fallback/index.js';
 import markdownVitePlugin from '../vite-plugin-markdown/index.js';
@@ -119,6 +120,7 @@ export async function createVite(
 			htmlVitePlugin(),
 			jsxVitePlugin({ settings, logging }),
 			astroPostprocessVitePlugin({ settings }),
+			mode === 'dev' && astroIntegrationsContainerPlugin({ settings, logging }),
 			astroScriptsPageSSRPlugin({ settings }),
 			astroHeadPlugin({ settings }),
 			astroScannerPlugin({ settings }),
diff --git a/packages/astro/src/core/dev/container.ts b/packages/astro/src/core/dev/container.ts
index cdc52c7e19ca..b65a2c48bbb6 100644
--- a/packages/astro/src/core/dev/container.ts
+++ b/packages/astro/src/core/dev/container.ts
@@ -8,7 +8,6 @@ import {
 	runHookConfigDone,
 	runHookConfigSetup,
 	runHookServerDone,
-	runHookServerSetup,
 	runHookServerStart,
 } from '../../integrations/index.js';
 import { createDefaultDevSettings, resolveRoot } from '../config/index.js';
@@ -91,7 +90,6 @@ export async function createContainer(params: CreateContainerParams = {}): Promi
 	);
 	await runHookConfigDone({ settings, logging });
 	const viteServer = await vite.createServer(viteConfig);
-	runHookServerSetup({ config: settings.config, server: viteServer, logging });
 
 	const container: Container = {
 		configFlag: params.configFlag,
diff --git a/packages/astro/src/vite-plugin-integrations-container/index.ts b/packages/astro/src/vite-plugin-integrations-container/index.ts
new file mode 100644
index 000000000000..0169a6f71560
--- /dev/null
+++ b/packages/astro/src/vite-plugin-integrations-container/index.ts
@@ -0,0 +1,20 @@
+import type { Plugin as VitePlugin } from 'vite';
+import type { AstroSettings } from '../@types/astro.js';
+import type { LogOptions } from '../core/logger/core.js';
+import { runHookServerSetup } from '../integrations/index.js';
+
+/** Connect Astro integrations into Vite, as needed. */
+export default function astroIntegrationsContainerPlugin({
+	settings,
+	logging,
+}: {
+	settings: AstroSettings;
+	logging: LogOptions;
+}): VitePlugin {
+	return {
+		name: 'astro:integration-container',
+		configureServer(server) {
+			runHookServerSetup({ config: settings.config, server, logging });
+		},
+	};
+}
diff --git a/packages/astro/test/fixtures/integration-server-setup/astro.config.mjs b/packages/astro/test/fixtures/integration-server-setup/astro.config.mjs
new file mode 100644
index 000000000000..0a0a336976b4
--- /dev/null
+++ b/packages/astro/test/fixtures/integration-server-setup/astro.config.mjs
@@ -0,0 +1,6 @@
+import { defineConfig } from 'rollup'
+import test from './integration.js'
+
+export default defineConfig({
+	integrations: [test()]
+})
diff --git a/packages/astro/test/fixtures/integration-server-setup/integration.js b/packages/astro/test/fixtures/integration-server-setup/integration.js
new file mode 100644
index 000000000000..bcbd86228a89
--- /dev/null
+++ b/packages/astro/test/fixtures/integration-server-setup/integration.js
@@ -0,0 +1,15 @@
+export default function() {
+	return {
+		name: '@astrojs/test-integration',
+		hooks: {
+			'astro:server:setup': ({ server }) => {
+				server.middlewares.use(
+					function middleware(req, res, next) {
+						res.setHeader('x-middleware', 'true');
+						next();
+					}
+				);
+			}
+		}
+	}
+}
diff --git a/packages/astro/test/fixtures/integration-server-setup/package.json b/packages/astro/test/fixtures/integration-server-setup/package.json
new file mode 100644
index 000000000000..c3ded67ef3a2
--- /dev/null
+++ b/packages/astro/test/fixtures/integration-server-setup/package.json
@@ -0,0 +1,9 @@
+{
+  "name": "@test/integration-server-setup",
+  "type": "module",
+  "version": "0.0.0",
+  "private": true,
+  "dependencies": {
+    "astro": "workspace:*"
+  }
+}
diff --git a/packages/astro/test/integration-server-setup.test.js b/packages/astro/test/integration-server-setup.test.js
new file mode 100644
index 000000000000..abe41d2bb8a3
--- /dev/null
+++ b/packages/astro/test/integration-server-setup.test.js
@@ -0,0 +1,24 @@
+import { expect } from 'chai';
+import { loadFixture } from './test-utils.js';
+
+describe('Integration server setup', () => {
+	/** @type {import('./test-utils').DevServer} */
+	let devServer;
+	/** @type {import('./test-utils').Fixture} */
+	let fixture;
+
+	before(async () => {
+		fixture = await loadFixture({ root: './fixtures/integration-server-setup/' });
+		devServer = await fixture.startDevServer();
+	});
+
+	after(async () => {
+		await devServer.stop();
+	});
+
+	it('Adds middlewares in dev', async () => {
+		const res = await fixture.fetch('/');
+
+		expect(res.headers.get('x-middleware')).to.equal('true');
+	});
+});
diff --git a/pnpm-lock.yaml b/pnpm-lock.yaml
index cfc4d3a50bb0..08c38a58a260 100644
--- a/pnpm-lock.yaml
+++ b/pnpm-lock.yaml
@@ -2139,6 +2139,12 @@ importers:
     dependencies:
       astro: link:../../..
 
+  packages/astro/test/fixtures/integration-server-setup:
+    specifiers:
+      astro: workspace:*
+    dependencies:
+      astro: link:../../..
+
   packages/astro/test/fixtures/jsx:
     specifiers:
       '@astrojs/preact': workspace:*