The pathogenesis of ulcerative colitis (UC) involves genetic, immunological, and environmental factors as well as gut microbiota dysbiosis. As a natural antioxidant with various pharmacological activities widely present in Oleaceae plants, oleuropein (OLE) exhibits anti-inflammatory, anti-tumor, antiviral, hypoglycemic, and cardioprotective effects. It has been validated that OLE extracted from olive oil can ameliorate UC. However, it remains unclear if and how OLE modulates the gut microbiota in the alleviation of UC. Therefore, this study was conducted to explore the mechanisms for OLE to alleviate UC induced by dextran sulfate sodium (DSS), with the focus placed on its regulatory function in the gut microbiota. The results indicated that OLE mitigated DSS-induced UC by enhancing the intestinal barrier function, reshaping the gut microbiota, and modulating bile acid metabolism. The fecal microbiota transplantation (FMT) experiment results further confirmed that the protective effect of OLE against UC could be mediated by alterations in the gut microbiota and their metabolites induced by OLE. Additionally, OLE increased the abundance of Lactobacillus and certain bile acid metabolites in the colon, including hyodeoxycholic acid (HDCA). HDCA could upregulate the expression of ZO-1 and claudin-3, restoring intestinal barrier integrity. Simultaneously, HDCA could inhibit the activation of the nuclear factor kappa-B (NF-κB) signaling pathway in the colon and relieve colonic inflammation. Overall, it was corroborated that OLE alleviated DSS-induced UC by modulating the gut microbiota and altering bile acid metabolism.