It has been reported that the ethanol extract of Wedelia chinensis attenuates murine colitis. Wedelolactone (WEL), a coumestane-type compound with many pharmacological activities, was isolated from W. chinensis. The present study aims to investigate the beneficial effects and underlying mechanisms of WEL on ulcerative colitis. In a dextran sodium sulfate (DSS)-induced mouse model, oral administration of WEL (50mg/kg) significantly attenuated pathological colonic damage and inhibited inflammatory infiltration, myeloperoxidase and alkaline phosphatase activities through MAPKs and NF-κB signaling pathways, while activating AMPK in colons treated with DSS. Further study revealed that WEL treatment dramatically inhibited NLRP3 inflammasome activation and caspase-1 phosphorylation to decrease IL-1β release in colons treated with DSS. In addition, WEL effectively regulates the disorder of skeleton proteins in colonic epithelial cells NCM460 exposed to TNF-α and protects the intestinal barrier function by activating AMPK in vivo. In summary, the AMPK-NLRP3-IL-1β signaling axis plays an important role in colitis following WEL treatments. These findings provide new insights into the pharmacological actions of WEL as a potential therapeutic agent for colitis.