diff --git a/lib/galaxy/dependencies/pinned-requirements.txt b/lib/galaxy/dependencies/pinned-requirements.txt
index 6beccd7eeae..76e56c6c8a1 100644
--- a/lib/galaxy/dependencies/pinned-requirements.txt
+++ b/lib/galaxy/dependencies/pinned-requirements.txt
@@ -102,7 +102,7 @@ griffelib==2.0.0
 groq==1.0.0
 grpcio==1.78.1
 grpcio-status==1.78.1
-gunicorn==25.1.0
+gunicorn==25.0.3
 gxformat2==0.22.0
 h11==0.16.0
 h5grove==3.0.0
diff --git a/packages/web_apps/setup.cfg b/packages/web_apps/setup.cfg
index 76625ee1ea5..fc633a6ff5e 100644
--- a/packages/web_apps/setup.cfg
+++ b/packages/web_apps/setup.cfg
@@ -44,7 +44,7 @@ install_requires =
     Babel
     CT3>=3.3.3
     fastapi>=0.124.0
-    gunicorn
+    gunicorn!=25.1.0
     httpx
     gxformat2
     Mako
diff --git a/packages/web_stack/setup.cfg b/packages/web_stack/setup.cfg
index 7532c6d525c..240eacdc1d0 100644
--- a/packages/web_stack/setup.cfg
+++ b/packages/web_stack/setup.cfg
@@ -41,7 +41,7 @@ python_requires = >=3.10
 [options.extras_require]
 test =
     pytest
-    gunicorn
+    gunicorn!=25.1.0
 
 [options.packages.find]
 exclude =
diff --git a/pyproject.toml b/pyproject.toml
index 3a27040e583..f4e889468db 100644
--- a/pyproject.toml
+++ b/pyproject.toml
@@ -42,7 +42,7 @@ dependencies = [
     "fs",
     "future>=1.0.0",  # Python 3.12 support
     "gravity>=1.2.0",  # Python 3.14 support
-    "gunicorn",
+    "gunicorn!=25.1.0",  # https://github.com/benoitc/gunicorn/discussions/3509
     "gxformat2>=0.21.0",
     "h5grove>=1.2.1",
     "h5py>=3.12",  # Python 3.13 support