diff --git a/src/_pytest/config/__init__.py b/src/_pytest/config/__init__.py
index 90099137c41..4a961b934cb 100644
--- a/src/_pytest/config/__init__.py
+++ b/src/_pytest/config/__init__.py
@@ -262,19 +262,6 @@ def _prepareconfig(
         raise
 
 
-def _fail_on_non_top_pytest_plugins(conftestpath, confcutdir):
-    msg = (
-        "Defining 'pytest_plugins' in a non-top-level conftest is no longer supported:\n"
-        "It affects the entire test suite instead of just below the conftest as expected.\n"
-        "  {}\n"
-        "Please move it to a top level conftest file at the rootdir:\n"
-        "  {}\n"
-        "For more information, visit:\n"
-        "  https://docs.pytest.org/en/latest/deprecations.html#pytest-plugins-in-non-top-level-conftest-files"
-    )
-    fail(msg.format(conftestpath, confcutdir), pytrace=False)
-
-
 class PytestPluginManager(PluginManager):
     """
     Overwrites :py:class:`pluggy.PluginManager <pluggy.PluginManager>` to add pytest-specific
@@ -499,12 +486,6 @@ def _importconftest(self, conftestpath):
                 _ensure_removed_sysmodule(conftestpath.purebasename)
             try:
                 mod = conftestpath.pyimport()
-                if (
-                    hasattr(mod, "pytest_plugins")
-                    and self._configured
-                    and not self._using_pyargs
-                ):
-                    _fail_on_non_top_pytest_plugins(conftestpath, self._confcutdir)
             except Exception:
                 raise ConftestImportFailure(conftestpath, sys.exc_info())
 
diff --git a/testing/test_config.py b/testing/test_config.py
index 7d827a46896..82a2330fcbf 100644
--- a/testing/test_config.py
+++ b/testing/test_config.py
@@ -1,4 +1,3 @@
-import os
 import re
 import sys
 import textwrap
@@ -1444,108 +1443,6 @@ def test_pytest_custom_cfg_unsupported(self, testdir):
             testdir.runpytest("-c", "custom.cfg")
 
 
-class TestPytestPluginsVariable:
-    def test_pytest_plugins_in_non_top_level_conftest_unsupported(self, testdir):
-        testdir.makepyfile(
-            **{
-                "subdirectory/conftest.py": """
-            pytest_plugins=['capture']
-        """
-            }
-        )
-        testdir.makepyfile(
-            """
-            def test_func():
-                pass
-        """
-        )
-        res = testdir.runpytest()
-        assert res.ret == 2
-        msg = "Defining 'pytest_plugins' in a non-top-level conftest is no longer supported"
-        res.stdout.fnmatch_lines(
-            [
-                "*{msg}*".format(msg=msg),
-                "*subdirectory{sep}conftest.py*".format(sep=os.sep),
-            ]
-        )
-
-    @pytest.mark.parametrize("use_pyargs", [True, False])
-    def test_pytest_plugins_in_non_top_level_conftest_unsupported_pyargs(
-        self, testdir, use_pyargs
-    ):
-        """When using --pyargs, do not emit the warning about non-top-level conftest warnings (#4039, #4044)"""
-
-        files = {
-            "src/pkg/__init__.py": "",
-            "src/pkg/conftest.py": "",
-            "src/pkg/test_root.py": "def test(): pass",
-            "src/pkg/sub/__init__.py": "",
-            "src/pkg/sub/conftest.py": "pytest_plugins=['capture']",
-            "src/pkg/sub/test_bar.py": "def test(): pass",
-        }
-        testdir.makepyfile(**files)
-        testdir.syspathinsert(testdir.tmpdir.join("src"))
-
-        args = ("--pyargs", "pkg") if use_pyargs else ()
-        res = testdir.runpytest(*args)
-        assert res.ret == (0 if use_pyargs else 2)
-        msg = (
-            msg
-        ) = "Defining 'pytest_plugins' in a non-top-level conftest is no longer supported"
-        if use_pyargs:
-            assert msg not in res.stdout.str()
-        else:
-            res.stdout.fnmatch_lines(["*{msg}*".format(msg=msg)])
-
-    def test_pytest_plugins_in_non_top_level_conftest_unsupported_no_top_level_conftest(
-        self, testdir
-    ):
-        subdirectory = testdir.tmpdir.join("subdirectory")
-        subdirectory.mkdir()
-        testdir.makeconftest(
-            """
-            pytest_plugins=['capture']
-        """
-        )
-        testdir.tmpdir.join("conftest.py").move(subdirectory.join("conftest.py"))
-
-        testdir.makepyfile(
-            """
-            def test_func():
-                pass
-        """
-        )
-
-        res = testdir.runpytest_subprocess()
-        assert res.ret == 2
-        msg = "Defining 'pytest_plugins' in a non-top-level conftest is no longer supported"
-        res.stdout.fnmatch_lines(
-            [
-                "*{msg}*".format(msg=msg),
-                "*subdirectory{sep}conftest.py*".format(sep=os.sep),
-            ]
-        )
-
-    def test_pytest_plugins_in_non_top_level_conftest_unsupported_no_false_positives(
-        self, testdir
-    ):
-        testdir.makepyfile(
-            "def test_func(): pass",
-            **{
-                "subdirectory/conftest": "pass",
-                "conftest": """
-                    import warnings
-                    warnings.filterwarnings('always', category=DeprecationWarning)
-                    pytest_plugins=['capture']
-                    """,
-            },
-        )
-        res = testdir.runpytest_subprocess()
-        assert res.ret == 0
-        msg = "Defining 'pytest_plugins' in a non-top-level conftest is no longer supported"
-        assert msg not in res.stdout.str()
-
-
 def test_report_implicit_args(testdir, monkeypatch):
     p = testdir.makepyfile(
         """