diff --git a/docs/changelog/3445.bugfix.rst b/docs/changelog/3445.bugfix.rst
new file mode 100644
index 0000000000..ad9329baac
--- /dev/null
+++ b/docs/changelog/3445.bugfix.rst
@@ -0,0 +1,2 @@
+Fix ``setenv`` modifications to ``PATH`` being overwritten when tox environment paths are set up - by
+:user:`Fridayai700`
diff --git a/src/tox/tox_env/api.py b/src/tox/tox_env/api.py
index a9cb3d8251..5a2c17f342 100644
--- a/src/tox/tox_env/api.py
+++ b/src/tox/tox_env/api.py
@@ -386,6 +386,9 @@ def environment_variables(self) -> dict[str, str]:
         result["PATH"] = self._make_path()
         for key in set_env:
             result[key] = set_env.load(key)
+        # if set_env modified PATH, re-prepend virtual-env paths (deduped) so they always come first
+        if self._paths and "PATH" in set_env:
+            result["PATH"] = self._make_path(result["PATH"])
         result["TOX_ENV_NAME"] = self.name
         result["TOX_WORK_DIR"] = str(self.core["work_dir"])
         result["TOX_ENV_DIR"] = str(self.conf["env_dir"])
@@ -406,11 +409,8 @@ def _paths(self) -> list[Path]:
     @_paths.setter
     def _paths(self, value: list[Path]) -> None:
         self._paths_private = value
-        # also update the environment variable with the new value
-        if self._env_vars is not None:  # pragma: no branch
-            # remove duplicates and prepend the tox env paths
-            result = self._make_path()
-            self._env_vars["PATH"] = result
+        # Invalidate cached env vars so they rebuild on next access, preserving set_env PATH modifications.
+        self._env_vars = None
 
     @property
     def _allow_externals(self) -> list[str]:
@@ -418,9 +418,9 @@ def _allow_externals(self) -> list[str]:
         result.extend(i.strip() for i in self.conf["allowlist_externals"])
         return result
 
-    def _make_path(self) -> str:
+    def _make_path(self, existing: str | None = None) -> str:
         values = dict.fromkeys(str(i) for i in self._paths)
-        values.update(dict.fromkeys(os.environ.get("PATH", "").split(os.pathsep)))
+        values.update(dict.fromkeys((existing or os.environ.get("PATH", "")).split(os.pathsep)))
         return os.pathsep.join(values)
 
     def execute(  # noqa: PLR0913
diff --git a/tests/tox_env/test_tox_env_api.py b/tests/tox_env/test_tox_env_api.py
index b83d5a811d..b3b857434b 100644
--- a/tests/tox_env/test_tox_env_api.py
+++ b/tests/tox_env/test_tox_env_api.py
@@ -174,3 +174,38 @@ def test_change_dir_is_relative_to_conf(tox_project: ToxProjectCreator) -> None:
     result.assert_success()
     lines = result.out.splitlines()
     assert lines[1] == f"change_dir = {prj.path / 'a'}"
+
+
+def test_setenv_path_not_overwritten(tox_project: ToxProjectCreator) -> None:
+    cmd = "import os; print(os.environ['PATH'])"
+    toml = f"""
+    [env_run_base]
+    package = "skip"
+    set_env.PATH = "{{env:PATH}}:/custom/test/path"
+    commands = [["python", "-c", "{cmd}"]]
+    """
+    project = tox_project({"tox.toml": toml})
+    result = project.run("r")
+    result.assert_success()
+    # The custom path from set_env must survive — not be overwritten
+    assert "/custom/test/path" in result.out
+
+
+def test_setenv_path_venv_paths_first(tox_project: ToxProjectCreator) -> None:
+    cmd = "import os; print(os.environ['PATH'])"
+    toml = f"""
+    [env_run_base]
+    package = "skip"
+    set_env.PATH = "{{env:PATH}}:/trailing/path"
+    commands = [["python", "-c", "{cmd}"]]
+    """
+    project = tox_project({"tox.toml": toml})
+    result = project.run("r")
+    result.assert_success()
+    path_line = next(line for line in result.out.splitlines() if "/trailing/path" in line)
+    path_entries = path_line.split(":")
+    # The virtual environment paths (containing .tox) must come before the trailing path
+    tox_idx = next((i for i, p in enumerate(path_entries) if ".tox" in p), None)
+    trailing_idx = next(i for i, p in enumerate(path_entries) if p == "/trailing/path")
+    assert tox_idx is not None, f"expected .tox path in PATH, got: {path_line}"
+    assert tox_idx < trailing_idx, f"venv paths should precede trailing path: {path_line}"