Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, driven by genetic, environmental, and immune system factors. However, its exact mechanisms remain unclear. Here, we demonstrate that CNPY2 plays a critical role in colitis by modulating macrophage activity. Mice with whole-body Cnpy2 knockout (KO) exhibited significantly reduced dextran sodium sulfate (DSS)-induced colitis compared to wild-type controls. Cnpy2 KO mice showed less mucosal barrier disruption and fewer lamina propria macrophages (LPMphis) following DSS treatment. Proinflammatory cytokine production was also diminished in the colons of Cnpy2 KO mice. Furthermore, Cnpy2 KO macrophages generated markedly lower levels of reactive oxygen species (ROS), partly through CHOP regulation. Notably, treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) completely abolished DSS-induced colitis in Cnpy2 KO mice. Thus, CNPY2 exacerbates DSS-induced colitis primarily through macrophage-specific effects, with ROS upregulation being central to its pathogenic role.