diff --git a/changelog/6646.bugfix.rst b/changelog/6646.bugfix.rst
new file mode 100644
index 000000000..4dba3ed07
--- /dev/null
+++ b/changelog/6646.bugfix.rst
@@ -0,0 +1 @@
+Assertion rewriting hooks are (re)stored for the current item, which fixes them being still used after e.g. pytester's :func:`testdir.runpytest <_pytest.pytester.Testdir.runpytest>` etc.
diff --git a/src/_pytest/assertion/__init__.py b/src/_pytest/assertion/__init__.py
index a060723a7..cdb034703 100644
--- a/src/_pytest/assertion/__init__.py
+++ b/src/_pytest/assertion/__init__.py
@@ -8,6 +8,7 @@ from _pytest.assertion import rewrite
 from _pytest.assertion import truncate
 from _pytest.assertion import util
 from _pytest.compat import TYPE_CHECKING
+from _pytest.config import hookimpl
 
 if TYPE_CHECKING:
     from _pytest.main import Session
@@ -105,7 +106,8 @@ def pytest_collection(session: "Session") -> None:
             assertstate.hook.set_session(session)
 
 
-def pytest_runtest_setup(item):
+@hookimpl(tryfirst=True, hookwrapper=True)
+def pytest_runtest_protocol(item):
     """Setup the pytest_assertrepr_compare and pytest_assertion_pass hooks
 
     The newinterpret and rewrite modules will use util._reprcompare if
@@ -143,6 +145,7 @@ def pytest_runtest_setup(item):
                 return res
         return None
 
+    saved_assert_hooks = util._reprcompare, util._assertion_pass
     util._reprcompare = callbinrepr
 
     if item.ihook.pytest_assertion_pass.get_hookimpls():
@@ -154,10 +157,9 @@ def pytest_runtest_setup(item):
 
         util._assertion_pass = call_assertion_pass_hook
 
+    yield
 
-def pytest_runtest_teardown(item):
-    util._reprcompare = None
-    util._assertion_pass = None
+    util._reprcompare, util._assertion_pass = saved_assert_hooks
 
 
 def pytest_sessionfinish(session):
diff --git a/src/_pytest/config/__init__.py b/src/_pytest/config/__init__.py
index ed3334e5f..d4477ba81 100644
--- a/src/_pytest/config/__init__.py
+++ b/src/_pytest/config/__init__.py
@@ -27,7 +27,6 @@ from pluggy import HookspecMarker
 from pluggy import PluginManager
 
 import _pytest._code
-import _pytest.assertion
 import _pytest.deprecated
 import _pytest.hookspec  # the extension point definitions
 from .exceptions import PrintHelp
@@ -260,6 +259,8 @@ class PytestPluginManager(PluginManager):
     """
 
     def __init__(self):
+        import _pytest.assertion
+
         super().__init__("pytest")
         # The objects are module objects, only used generically.
         self._conftest_plugins = set()  # type: Set[object]
@@ -891,6 +892,8 @@ class Config:
         ns, unknown_args = self._parser.parse_known_and_unknown_args(args)
         mode = getattr(ns, "assertmode", "plain")
         if mode == "rewrite":
+            import _pytest.assertion
+
             try:
                 hook = _pytest.assertion.install_importhook(self)
             except SystemError:
diff --git a/testing/test_assertion.py b/testing/test_assertion.py
index e975a3fea..dc260b39f 100644
--- a/testing/test_assertion.py
+++ b/testing/test_assertion.py
@@ -72,10 +72,19 @@ class TestImportHookInstallation:
         result = testdir.runpytest_subprocess()
         result.stdout.fnmatch_lines(
             [
-                "E * AssertionError: ([[][]], [[][]], [[]<TestReport *>[]])*",
-                "E * assert"
-                " {'failed': 1, 'passed': 0, 'skipped': 0} =="
-                " {'failed': 0, 'passed': 1, 'skipped': 0}",
+                ">       r.assertoutcome(passed=1)",
+                "E       AssertionError: ([[][]], [[][]], [[]<TestReport *>[]])*",
+                "E       assert {'failed': 1,... 'skipped': 0} == {'failed': 0,... 'skipped': 0}",
+                "E         Omitting 1 identical items, use -vv to show",
+                "E         Differing items:",
+                "E         Use -v to get the full diff",
+            ]
+        )
+        # XXX: unstable output.
+        result.stdout.fnmatch_lines_random(
+            [
+                "E         {'failed': 1} != {'failed': 0}",
+                "E         {'passed': 0} != {'passed': 1}",
             ]
         )