diff --git a/picard/__init__.py b/picard/__init__.py
index a99c439046..eb50228dc6 100644
--- a/picard/__init__.py
+++ b/picard/__init__.py
@@ -41,7 +41,7 @@
 PICARD_DISPLAY_NAME = "MusicBrainz Picard"
 PICARD_APP_ID = "org.musicbrainz.Picard"
 PICARD_DESKTOP_NAME = PICARD_APP_ID + ".desktop"
-PICARD_VERSION = Version(3, 0, 0, 'dev', 4)
+PICARD_VERSION = Version(3, 0, 0, 'dev', 5)
 
 
 # optional build version
diff --git a/picard/config_upgrade.py b/picard/config_upgrade.py
index ee0b7685ce..8627b99d3a 100644
--- a/picard/config_upgrade.py
+++ b/picard/config_upgrade.py
@@ -31,6 +31,7 @@
     getmembers,
     isfunction,
 )
+import os
 import re
 import sys
 
@@ -47,6 +48,7 @@
 )
 from picard.const.defaults import (
     DEFAULT_FILE_NAMING_FORMAT,
+    DEFAULT_REPLACEMENT,
     DEFAULT_SCRIPT_NAME,
 )
 from picard.const.sys import IS_FROZEN
@@ -550,6 +552,15 @@ def upgrade_to_v3_0_0dev4(config):
         config.persist.remove('file_view_header_state')
 
 
+def upgrade_to_v3_0_0dev5(config):
+    """Ensure "replace_dir_separator" contains no directory separator"""
+    replace_dir_separator = config.setting['replace_dir_separator']
+    replace_dir_separator = replace_dir_separator.replace(os.sep, DEFAULT_REPLACEMENT)
+    if os.altsep:
+        replace_dir_separator = replace_dir_separator.replace(os.altsep, DEFAULT_REPLACEMENT)
+    config.setting['replace_dir_separator'] = replace_dir_separator
+
+
 def rename_option(config, old_opt, new_opt, option_type, default):
     _s = config.setting
     if old_opt in _s:
diff --git a/picard/ui/options/renaming_compat.py b/picard/ui/options/renaming_compat.py
index d204c2a044..9a386b3b7c 100644
--- a/picard/ui/options/renaming_compat.py
+++ b/picard/ui/options/renaming_compat.py
@@ -4,7 +4,7 @@
 #
 # Copyright (C) 2006-2008, 2011 Lukáš Lalinský
 # Copyright (C) 2008-2009 Nikolai Prokoschenko
-# Copyright (C) 2009-2010, 2014-2015, 2018-2022 Philipp Wolfer
+# Copyright (C) 2009-2010, 2014-2015, 2018-2024 Philipp Wolfer
 # Copyright (C) 2011-2013 Michael Wiencek
 # Copyright (C) 2011-2013 Wieland Hoffmann
 # Copyright (C) 2013 Calvin Walton
@@ -32,7 +32,7 @@
 # along with this program; if not, write to the Free Software
 # Foundation, Inc., 51 Franklin Street, Fifth Floor, Boston, MA 02110-1301, USA.
 
-
+import os
 import re
 
 from PyQt6 import (
@@ -56,7 +56,10 @@
     Ui_RenamingCompatOptionsPage,
 )
 from picard.ui.forms.ui_win_compat_dialog import Ui_WinCompatDialog
-from picard.ui.options import OptionsPage
+from picard.ui.options import (
+    OptionsCheckError,
+    OptionsPage,
+)
 
 
 class RenamingCompatOptionsPage(OptionsPage):
@@ -80,6 +83,7 @@ def __init__(self, parent=None):
         self.ui.windows_long_paths.toggled.connect(self.on_options_changed)
         self.ui.replace_spaces_with_underscores.toggled.connect(self.on_options_changed)
         self.ui.replace_dir_separator.textChanged.connect(self.on_options_changed)
+        self.ui.replace_dir_separator.setValidator(NoDirectorySeparatorValidator())
         self.ui.btn_windows_compatibility_change.clicked.connect(self.open_win_compat_dialog)
 
         self.register_setting('ascii_filenames', ['ascii_filenames'])
@@ -113,6 +117,14 @@ def save(self):
         for key, value in options.items():
             config.setting[key] = value
 
+    def check(self):
+        (valid_state, _text, _pos) = self.ui.replace_dir_separator.validator().validate(self.ui.replace_dir_separator.text(), 0)
+        if valid_state != QtGui.QValidator.State.Acceptable:
+            raise OptionsCheckError(
+                _("Invalid directory separator replacement"),
+                _("The replacement for directory separators must not be itself a directory separator.")
+            )
+
     def toggle_windows_long_paths(self, state):
         if state and not system_supports_long_paths():
             dialog = QtWidgets.QMessageBox(
@@ -147,6 +159,15 @@ def open_win_compat_dialog(self):
             self.on_options_changed()
 
 
+class NoDirectorySeparatorValidator(QtGui.QValidator):
+    def validate(self, text: str, pos):
+        if os.sep in text or (os.altsep and os.altsep in text):
+            state = QtGui.QValidator.State.Invalid
+        else:
+            state = QtGui.QValidator.State.Acceptable
+        return state, text, pos
+
+
 class WinCompatReplacementValidator(QtGui.QValidator):
     _re_valid_win_replacement = re.compile(r'^[^"*:<>?|/\\\s]?$')
 
diff --git a/test/test_config_upgrade.py b/test/test_config_upgrade.py
index 66e7e8330c..3078f64ae9 100644
--- a/test/test_config_upgrade.py
+++ b/test/test_config_upgrade.py
@@ -21,6 +21,7 @@
 # along with this program; if not, write to the Free Software
 # Foundation, Inc., 51 Franklin Street, Fifth Floor, Boston, MA 02110-1301, USA.
 
+import os
 
 from PyQt6.QtCore import QByteArray
 
@@ -66,9 +67,11 @@
     upgrade_to_v2_8_0dev2,
     upgrade_to_v3_0_0dev3,
     upgrade_to_v3_0_0dev4,
+    upgrade_to_v3_0_0dev5,
 )
 from picard.const.defaults import (
     DEFAULT_FILE_NAMING_FORMAT,
+    DEFAULT_REPLACEMENT,
     DEFAULT_SCRIPT_NAME,
 )
 from picard.util import unique_numbered_title
@@ -541,3 +544,14 @@ def test_upgrade_to_v3_0_0dev4(self):
         upgrade_to_v3_0_0dev4(self.config)
         self.assertEqual(b'', self.config.persist['album_view_header_state'])
         self.assertEqual(b'', self.config.persist['file_view_header_state'])
+
+    def test_upgrade_to_v3_0_0dev5(self):
+        TextOption('setting', 'replace_dir_separator', DEFAULT_REPLACEMENT)
+        self.config.setting['replace_dir_separator'] = os.sep
+        upgrade_to_v3_0_0dev5(self.config)
+        self.assertEqual(DEFAULT_REPLACEMENT, self.config.setting['replace_dir_separator'])
+
+        if os.altsep:
+            self.config.setting['replace_dir_separator'] = os.altsep
+            upgrade_to_v3_0_0dev5(self.config)
+            self.assertEqual(DEFAULT_REPLACEMENT, self.config.setting['replace_dir_separator'])