Ulcerative colitis (UC) is a chronic inflammatory disease. Intestinal mucosal injury is a significant factor in UC. Pyroptosis is a kind of programmed cell death induced by inflammatory caspases. Proteasome 20S subunit beta 5 (PSMB5) promotes cell viability. The purpose of this study was to determine the impact of PSMB5 on intestinal mucosal injury and to elucidate the underlying processes in dextran sulfate sodium- (DSS-) induced UC mice. Kunming (KM) mice received 3% DSS for 5 days to induce UC. We collected clinical symptoms, body weight, colon length, and histological changes. MDA (malondialdehyde) and SOD (superoxide dismutase) levels were determined using an ELISA assay. RT-PCR was used to assess the expression of IL-1 β and IL-18. PSMB5 demonstrated a significant effect against UC by increasing body weight and colon length and decreasing DAI (disease activity index), colon macroscopic damage index (CMDI), histological injury scores, and reactive oxygen species (ROS), MDA, and SOD levels, thereby alleviating histopathological changes and inhibiting oxidative stress. HIEC-6 cells were exposed to lipopolysaccharide (LPS) condition with or without PSMB5, along with caspase-1 inhibitor (Z-VAD-FMK), NLRP3 inhibitor (MCC950), and ROS scavenger N-acetylcysteine (NAC). The viability of the cells, the release of lactate dehydrogenase (LDH), and intracellular ROS generation were determined using assay kits. Western blot analysis was used to determine the levels of NLRP3, ASC, cleaved caspase-1 (p20), pro-IL-1 β , IL-1 β , pro-IL-18, and IL-18. PSMB5 overexpression enhanced the inflammatory damage in LPS-treated HIEC-6 cells by activating the NLRP3 inflammasome and mediating pyroptosis, as demonstrated by increased LDH release and lower cell viability, as well as increased expression of NLRP3, ASC, cleaved caspase-1 (p20), IL-1, and IL-18. Meanwhile, NAC protected HIEC-6 cells from LPS-induced damage by reversing the activation of the NLRP3 inflammasome-mediated pyroptosis. In conclusion, PSMB5 may lower HIEC-6 cell susceptibility to LPS and ameliorate UC-induced HIEC-6 cell damage by decreasing ROS generation and hence inhibiting NLRP3-mediated pyroptosis.