diff --git a/django/db/models/query.py b/django/db/models/query.py
index c943cfe219..f88662de12 100644
--- a/django/db/models/query.py
+++ b/django/db/models/query.py
@@ -33,7 +33,6 @@ from django.db.models.utils import (
     resolve_callables,
 )
 from django.utils import timezone
-from django.utils.deprecation import RemovedInDjango50Warning
 from django.utils.functional import cached_property, partition
 
 # The maximum number of results to fetch in a get() query.
@@ -529,16 +528,9 @@ class QuerySet(AltersData):
         """
         if chunk_size is None:
             if self._prefetch_related_lookups:
-                # When the deprecation ends, replace with:
-                # raise ValueError(
-                #     'chunk_size must be provided when using '
-                #     'QuerySet.iterator() after prefetch_related().'
-                # )
-                warnings.warn(
-                    "Using QuerySet.iterator() after prefetch_related() "
-                    "without specifying chunk_size is deprecated.",
-                    category=RemovedInDjango50Warning,
-                    stacklevel=2,
+                raise ValueError(
+                    "chunk_size must be provided when using QuerySet.iterator() after "
+                    "prefetch_related()."
                 )
         elif chunk_size <= 0:
             raise ValueError("Chunk size must be strictly positive.")
diff --git a/docs/ref/models/querysets.txt b/docs/ref/models/querysets.txt
index 4a1836ba14..df77cabe33 100644
--- a/docs/ref/models/querysets.txt
+++ b/docs/ref/models/querysets.txt
@@ -2476,12 +2476,6 @@ value for ``chunk_size`` will result in Django using an implicit default of
 Depending on the database backend, query results will either be loaded all at
 once or streamed from the database using server-side cursors.
 
-.. deprecated:: 4.1
-
-    Using ``iterator()`` on a queryset that prefetches related objects without
-    providing the ``chunk_size`` is deprecated. In Django 5.0, an exception
-    will be raise.
-
 With server-side cursors
 ^^^^^^^^^^^^^^^^^^^^^^^^
 
diff --git a/docs/releases/5.0.txt b/docs/releases/5.0.txt
index d39b8d314a..e058f4a76c 100644
--- a/docs/releases/5.0.txt
+++ b/docs/releases/5.0.txt
@@ -319,3 +319,6 @@ to remove usage of these features.
   ``SimpleTestCase.assertFormError()`` and ``assertFormsetError()`` is removed.
 
 * ``django.contrib.sessions.serializers.PickleSerializer`` is removed.
+
+* The usage of ``QuerySet.iterator()`` on a queryset that prefetches related
+  objects without providing the ``chunk_size`` argument is no longer allowed.
diff --git a/tests/prefetch_related/tests.py b/tests/prefetch_related/tests.py
index c153c0d9ec..c8b9a9538b 100644
--- a/tests/prefetch_related/tests.py
+++ b/tests/prefetch_related/tests.py
@@ -12,8 +12,7 @@ from django.test import (
     skipIfDBFeature,
     skipUnlessDBFeature,
 )
-from django.test.utils import CaptureQueriesContext, ignore_warnings
-from django.utils.deprecation import RemovedInDjango50Warning
+from django.test.utils import CaptureQueriesContext
 
 from .models import (
     Article,
@@ -385,25 +384,12 @@ class PrefetchRelatedTests(TestDataMixin, TestCase):
             [self.author1, self.author1, self.author3, self.author4],
         )
 
-    @ignore_warnings(category=RemovedInDjango50Warning)
-    def test_m2m_prefetching_iterator_without_chunks(self):
-        # prefetch_related() is ignored.
-        with self.assertNumQueries(5):
-            authors = [
-                b.authors.first()
-                for b in Book.objects.prefetch_related("authors").iterator()
-            ]
-        self.assertEqual(
-            authors,
-            [self.author1, self.author1, self.author3, self.author4],
-        )
-
-    def test_m2m_prefetching_iterator_without_chunks_warning(self):
+    def test_m2m_prefetching_iterator_without_chunks_error(self):
         msg = (
-            "Using QuerySet.iterator() after prefetch_related() without "
-            "specifying chunk_size is deprecated."
+            "chunk_size must be provided when using QuerySet.iterator() after "
+            "prefetch_related()."
         )
-        with self.assertWarnsMessage(RemovedInDjango50Warning, msg):
+        with self.assertRaisesMessage(ValueError, msg):
             Book.objects.prefetch_related("authors").iterator()