diff --git a/aws_lambda_powertools/event_handler/api_gateway.py b/aws_lambda_powertools/event_handler/api_gateway.py
index 37e2265ea8a..0ddf287f264 100644
--- a/aws_lambda_powertools/event_handler/api_gateway.py
+++ b/aws_lambda_powertools/event_handler/api_gateway.py
@@ -2008,6 +2008,7 @@ def include_router(self, router: "Router", prefix: Optional[str] = None) -> None
         # use pointer to allow context clearance after event is processed e.g., resolve(evt, ctx)
         router.context = self.context
 
+        # Iterate through the routes defined in the router to configure and apply middlewares for each route
         for route, func in router._routes.items():
             new_route = route
 
@@ -2017,7 +2018,8 @@ def include_router(self, router: "Router", prefix: Optional[str] = None) -> None
                 new_route = (rule, *route[1:])
 
             # Middlewares are stored by route separately - must grab them to include
-            middlewares = router._routes_with_middleware.get(new_route)
+            # Middleware store the route without prefix, so we must not include prefix when grabbing
+            middlewares = router._routes_with_middleware.get(route)
 
             # Need to use "type: ignore" here since mypy does not like a named parameter after
             # tuple expansion since may cause duplicate named parameters in the function signature.
diff --git a/tests/functional/event_handler/test_api_gateway.py b/tests/functional/event_handler/test_api_gateway.py
index 18d10ec4167..8ad5ac35b18 100644
--- a/tests/functional/event_handler/test_api_gateway.py
+++ b/tests/functional/event_handler/test_api_gateway.py
@@ -1197,6 +1197,25 @@ def base():
     assert result["multiValueHeaders"]["Content-Type"] == [content_types.APPLICATION_JSON]
 
 
+def test_api_gateway_app_with_strip_prefix_and_route_prefix():
+    # GIVEN all routes are stripped from its version e.g., /v1
+    app = ApiGatewayResolver(strip_prefixes=["/v1"])
+    router = Router()
+
+    event = {"httpMethod": "GET", "path": "/v1/users/leandro", "resource": "/users"}
+
+    @router.get("<user_id>")
+    def base(user_id: str):
+        return {"user": user_id}
+
+    # WHEN a router is included prefixing all routes with "/users/"
+    app.include_router(router, prefix="/users/")
+    result = app(event, {})
+
+    # THEN route correctly to the registered route after stripping each prefix (global + router)
+    assert result["statusCode"] == 200
+
+
 def test_api_gateway_app_router():
     # GIVEN a Router with registered routes
     app = ApiGatewayResolver()
diff --git a/tests/functional/event_handler/test_api_middlewares.py b/tests/functional/event_handler/test_api_middlewares.py
index 8f98b93343f..58bec259072 100644
--- a/tests/functional/event_handler/test_api_middlewares.py
+++ b/tests/functional/event_handler/test_api_middlewares.py
@@ -397,6 +397,34 @@ def dummy_route():
     assert result["statusCode"] == 200
 
 
+def test_api_gateway_middleware_with_include_router_prefix():
+    # GIVEN an App and Router instance
+    app = ApiGatewayResolver()
+    router = Router()
+
+    def app_middleware(app: EventHandlerInstance, next_middleware: NextMiddleware):
+        # AND a variable injected into resolver context
+        app.append_context(injected="injected_value")
+        return next_middleware(app)
+
+    # WHEN we register a route with a middleware
+    @router.get("/path", middlewares=[app_middleware])
+    def dummy_route():
+        # THEN we should have access to the middleware's injected variable
+        assert app.context["injected"] == "injected_value"
+
+        return Response(status_code=200, body="works!")
+
+    # WHEN register the route with a prefix
+    app.include_router(router, prefix="/my")
+
+    # THEN resolving a request must execute the middleware
+    # and return a successful response http 200 status code
+    result = app(API_REST_EVENT, {})
+
+    assert result["statusCode"] == 200
+
+
 @pytest.mark.parametrize(
     "app, event",
     [