diff --git a/src/_pytest/pytester.py b/src/_pytest/pytester.py
index 2c6ff20208e..f936074d61e 100644
--- a/src/_pytest/pytester.py
+++ b/src/_pytest/pytester.py
@@ -795,6 +795,14 @@ def inline_genitems(self, *args):
         items = [x.item for x in rec.getcalls("pytest_itemcollected")]
         return items, rec
 
+    def _get_isolated_env(self):
+        tmpdir = str(self.tmpdir)
+        return (
+            # Do not load user config.
+            ("HOME", tmpdir),
+            ("USERPROFILE", tmpdir),
+        )
+
     def inline_run(self, *args, **kwargs):
         """Run ``pytest.main()`` in-process, returning a HookRecorder.
 
@@ -819,8 +827,8 @@ def inline_run(self, *args, **kwargs):
         try:
             # Do not load user config (during runs only).
             mp_run = MonkeyPatch()
-            mp_run.setenv("HOME", str(self.tmpdir))
-            mp_run.setenv("USERPROFILE", str(self.tmpdir))
+            for k, v in self._get_isolated_env():
+                mp_run.setenv(k, v)
             finalizers.append(mp_run.undo)
 
             # When running pytest inline any plugins active in the main test
@@ -1050,20 +1058,33 @@ def popen(
     ):
         """Invoke subprocess.Popen.
 
-        This calls subprocess.Popen making sure the current working directory
-        is in the PYTHONPATH.
+        This calls subprocess.Popen, making sure the current working directory
+        is in the PYTHONPATH by default.
 
-        You probably want to use :py:meth:`run` instead.
+        Optional keyword arguments:
+
+        :param env: OS environment to be used as is (no PYTHONPATH adjustment,
+                    nor isolation for HOME etc).
+        :param env_update: OS environment values to update the current
+                           environment with.
+                           PYTHONPATH gets adjusted if not passed in explicitly.
 
+        You probably want to use :py:meth:`run` instead.
         """
-        env = os.environ.copy()
-        env["PYTHONPATH"] = os.pathsep.join(
-            filter(None, [os.getcwd(), env.get("PYTHONPATH", "")])
-        )
-        # Do not load user config.
-        env["HOME"] = str(self.tmpdir)
-        env["USERPROFILE"] = env["HOME"]
-        kw["env"] = env
+        if "env" in kw:
+            env = kw.pop("env")
+            if "env_update" in kw:
+                raise ValueError("env and env_update are mutually exclusive")
+        else:
+            env = os.environ.copy()
+            env.update(self._get_isolated_env())
+
+            env_update = kw.pop("env_update", {})
+            if "PYTHONPATH" not in env_update:
+                env["PYTHONPATH"] = os.pathsep.join(
+                    filter(None, [os.getcwd(), env.get("PYTHONPATH", "")])
+                )
+            env.update(env_update)
 
         if stdin is Testdir.CLOSE_STDIN:
             kw["stdin"] = subprocess.PIPE
@@ -1072,7 +1093,7 @@ def popen(
         else:
             kw["stdin"] = stdin
 
-        popen = subprocess.Popen(cmdargs, stdout=stdout, stderr=stderr, **kw)
+        popen = subprocess.Popen(cmdargs, stdout=stdout, stderr=stderr, env=env, **kw)
         if stdin is Testdir.CLOSE_STDIN:
             popen.stdin.close()
         elif isinstance(stdin, bytes):
diff --git a/testing/test_pytester.py b/testing/test_pytester.py
index b76d413b7b1..02d52479267 100644
--- a/testing/test_pytester.py
+++ b/testing/test_pytester.py
@@ -22,6 +22,11 @@
 from _pytest.pytester import SysModulesSnapshot
 from _pytest.pytester import SysPathsSnapshot
 
+try:
+    import mock
+except ImportError:
+    import unittest.mock as mock
+
 
 def test_make_hook_recorder(testdir):
     item = testdir.getitem("def test_func(): pass")
@@ -559,3 +564,36 @@ def test_popen_default_stdin_stderr_and_stdin_None(testdir):
     assert stdout.splitlines() == [b"", b"stdout"]
     assert stderr.splitlines() == [b"stderr"]
     assert proc.returncode == 0
+
+
+def test_popen_env(testdir, monkeypatch):
+    monkeypatch.delenv("PYTHONPATH", raising=False)
+    popen_args = (["cmd"], None, None)
+
+    with mock.patch("subprocess.Popen") as m:
+        testdir.popen(*popen_args)
+        env = m.call_args[1]["env"]
+        assert set(env.keys()) == set(
+            list(os.environ.keys()) + ["PYTHONPATH", "USERPROFILE", "HOME"]
+        )
+        assert env["PYTHONPATH"] == os.getcwd()
+
+        # Updates PYTHONPATH by default.
+        monkeypatch.setenv("PYTHONPATH", "custom")
+        testdir.popen(*popen_args)
+        env = m.call_args[1]["env"]
+        assert env["PYTHONPATH"] == os.pathsep.join((os.getcwd(), "custom"))
+
+        # Uses explicit PYTHONPATH via env_update.
+        testdir.popen(*popen_args, env_update={"PYTHONPATH": "mypp", "CUSTOM_ENV": "1"})
+        env = m.call_args[1]["env"]
+        assert env["PYTHONPATH"] == "mypp"
+        assert env["CUSTOM_ENV"] == "1"
+
+        # Uses explicit env only.
+        testdir.popen(*popen_args, env={"CUSTOM_ENV": "1"})
+        env = m.call_args[1]["env"]
+        assert env == {"CUSTOM_ENV": "1"}
+
+    with pytest.raises(ValueError, match="env and env_update are mutually exclusive"):
+        testdir.popen(*popen_args, env={}, env_update={})