Intestinal microbiota imbalance may worsen the progression of ulcerative colitis (UC). Lacticaseibacillus rhamnosus LS8 (LR) has the potential ability to regulate microbiota through producing a novel antibacterial substance, cyclic opine: cycloalanopine. This study aimed to investigate whether LR could ameliorate dextran sulfate sodium-induced UC in mice via modulating intestinal microbiota using fecal microbiota transplantation (FMT) experiment. The results showed that both LR and FMT attenuated UC as evidenced by 1) alleviating disease activity index and colonic pathology; 2) up-regulating MUCs and tight junction proteins; 3) increasing oxidative mediators and decreasing antioxidant mediators; 4) down-regulating proinflammatory cytokines and chemokines. These results were mainly attributable to the microbiota-regulating effect of LR, including increasing beneficial bacteria (like Akkermansia) and its related SCFAs, while decreasing harmful bacteria (like Proteobacteria) and its related LPS, thereby suppressing the hyperactivation of TLR4/NF-κB pathway. Consequently, LR can alleviate UC and is a potential dietary supplement to attenuate UC.