diff --git a/changelog/4913.trivial.rst b/changelog/4913.trivial.rst
new file mode 100644
index 000000000..7846775cc
--- /dev/null
+++ b/changelog/4913.trivial.rst
@@ -0,0 +1 @@
+Fix pytest tests invocation with custom ``PYTHONPATH``.
diff --git a/testing/test_collection.py b/testing/test_collection.py
index 97c46d8c2..37f7ad89c 100644
--- a/testing/test_collection.py
+++ b/testing/test_collection.py
@@ -2,6 +2,7 @@ from __future__ import absolute_import
 from __future__ import division
 from __future__ import print_function
 
+import os
 import pprint
 import sys
 import textwrap
@@ -1108,7 +1109,7 @@ def test_collect_pyargs_with_testpaths(testdir, monkeypatch):
     """
         )
     )
-    monkeypatch.setenv("PYTHONPATH", str(testdir.tmpdir))
+    monkeypatch.setenv("PYTHONPATH", str(testdir.tmpdir), prepend=os.pathsep)
     with root.as_cwd():
         result = testdir.runpytest_subprocess()
     result.stdout.fnmatch_lines(["*1 passed in*"])