diff --git a/nixos/modules/system/boot/stage-2-init.sh b/nixos/modules/system/boot/stage-2-init.sh
index b5627ec8e5717..fa2884d087081 100755
--- a/nixos/modules/system/boot/stage-2-init.sh
+++ b/nixos/modules/system/boot/stage-2-init.sh
@@ -69,7 +69,8 @@ fi
 chown -f 0:30000 /nix/store
 chmod -f 1775 /nix/store
 if [ -n "@readOnlyNixStore@" ]; then
-    if ! [[ "$(findmnt --noheadings --output OPTIONS /nix/store)" =~ ro(,|$) ]]; then
+    # #375257: Ensure that we pick the "top" (i.e. last) mount so we don't get a false positive for a lower mount.
+    if ! [[ "$(findmnt --direction backward --first-only --noheadings --output OPTIONS /nix/store)" =~ (^|,)ro(,|$) ]]; then
         if [ -z "$container" ]; then
             mount --bind /nix/store /nix/store
         else
diff --git a/nixos/tests/all-tests.nix b/nixos/tests/all-tests.nix
index 2255c2f5eacc0..cb7f599eb6eaf 100644
--- a/nixos/tests/all-tests.nix
+++ b/nixos/tests/all-tests.nix
@@ -165,6 +165,7 @@ in {
   boot = handleTestOn ["x86_64-linux" "aarch64-linux"] ./boot.nix {};
   bootspec = handleTestOn ["x86_64-linux"] ./bootspec.nix {};
   boot-stage1 = handleTest ./boot-stage1.nix {};
+  boot-stage2 = handleTest ./boot-stage2.nix {};
   borgbackup = handleTest ./borgbackup.nix {};
   borgmatic = handleTest ./borgmatic.nix {};
   botamusique = handleTest ./botamusique.nix {};
diff --git a/nixos/tests/boot-stage2.nix b/nixos/tests/boot-stage2.nix
new file mode 100644
index 0000000000000..90f0ac01207c0
--- /dev/null
+++ b/nixos/tests/boot-stage2.nix
@@ -0,0 +1,73 @@
+import ./make-test-python.nix (
+  { pkgs, ... }:
+  {
+    name = "boot-stage2";
+
+    nodes.machine =
+      {
+        config,
+        pkgs,
+        lib,
+        ...
+      }:
+      {
+        virtualisation = {
+          emptyDiskImages = [ 256 ];
+
+          # Mount an ext4 as the upper layer of the Nix store.
+          fileSystems = {
+            "/nix/store" = lib.mkForce {
+              device = "/dev/vdb"; # the above disk image
+              fsType = "ext4";
+
+              # data=journal always displays after errors=remount-ro; this is only needed because of the overlay
+              # and #375257 will trigger with `errors=remount-ro` on a non-overlaid store:
+              # see ordering in https://github.com/torvalds/linux/blob/v6.12/fs/ext4/super.c#L2974
+              options = [
+                "defaults"
+                "errors=remount-ro"
+                "data=journal"
+              ];
+            };
+          };
+        };
+
+        boot = {
+          initrd = {
+            # Format the upper Nix store.
+            postDeviceCommands = ''
+              ${pkgs.e2fsprogs}/bin/mkfs.ext4 /dev/vdb
+            '';
+
+            # Overlay the RO store onto it.
+            # Note that bug #375257 can be triggered without an overlay,
+            # using the errors=remount-ro option (or similar) or with an overlay where any of the
+            # paths ends in 'ro'. The offending mountpoint also has to be the last (top) one
+            # if an option ending in 'ro' is the last in the list, so test both cases here.
+            postMountCommands = ''
+              mkdir -p /mnt-root/nix/store/ro /mnt-root/nix/store/rw /mnt-root/nix/store/work
+              mount --bind /mnt-root/nix/.ro-store /mnt-root/nix/store/ro
+              mount -t overlay overlay \
+                -o lowerdir=/mnt-root/nix/store/ro,upperdir=/mnt-root/nix/store/rw,workdir=/mnt-root/nix/store/work \
+                /mnt-root/nix/store
+            '';
+
+            kernelModules = [ "overlay" ];
+          };
+
+          postBootCommands = ''
+            touch /etc/post-boot-ran
+            mount
+          '';
+        };
+      };
+
+    testScript = ''
+      machine.wait_for_unit("multi-user.target")
+      machine.succeed("test /etc/post-boot-ran")
+      machine.fail("touch /nix/store/should-not-work");
+    '';
+
+    meta.maintainers = with pkgs.lib.maintainers; [ numinit ];
+  }
+)