Inflammatory bowel disease (IBD) is associated with gut dysbiosis and can lead to colitis-associated malignancies. Bacteroides uniformis (Bu) regulates animal intestinal homeostasis; however, the mechanism by which it alleviates colitis in mice remains unknown. We investigated the effects of B. uniformis JCM5828 and its metabolites on female C57BL/6J mice with dextran sulfate sodium salt (DSS) induced colitis. Treatment with Bu considerably alleviated colitis progression and restored the mechanical and immune barrier protein expression. Additionally, Bu increased the abundance of the symbiotic bacteria Bifidobacterium and Lactobacillus vaginalis while decreasing that of pathogenic Escherichia-Shigella, and modulated intestinal bile acid metabolism. Bu largely regulated the expression of key regulatory proteins of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways in colonic tissues and the differentiation of TH17 cells. However, Bu could not directly inhibit TH17 cell differentiation in vitro; it modulated the process in the lamina propria by participating in bile acid metabolism and regulating key metabolites (alpha-muricholic, hyodeoxycholic, and isolithocholic acid), thereby modulating the intestinal immune response. Our findings suggest that Bu or bile acid supplements are potential therapies for colitis and other diseases associated with intestinal barrier dysfunction.