Ulcerative colitis (UC) is a chronic intestinal inflammatory disease characterized by a complex pathogenesis. Weizmannia coagulans has emerged as a potential probiotic for treating intestinal disorders. This study aimed to assess the therapeutic impact of W. coagulans BC99 on mice with DSS-induced UC and to elucidate its underlying mechanism of action. Our findings revealed that BC99 administration ameliorated symptoms associated with DSS-induced UC mice, as evidenced by reduced disease activity indexes, reversal of weight loss, and normalization of colon length. Furthermore, BC99 treatment also protected the integrity of the intestinal barrier through maintaining the antioxidant activity and the expression of tight junction proteins (ZO-1 and occludin), and regulating the inflammatory cytokines in DSS-induced UC mice. Additionally, BC99 supplementation enhanced the production of short-chain fatty acids (SCFAs) through the proliferation of SCFA-producing bacteria, including Bidobacterium, Blautia and Faecallbaculum. Notably, the NF-κB signaling pathway was found to be closely related to BC99 treatment in DSS-induced UC mice. The positive protein expression and the mRNA expression of TLR4, MyD88 and p65 in colon tissue were all detected in BC99-treated groups, which indicating that BC99 could alleviate UC symptoms by inhibiting TLR4/MyD88/ NF-κB signaling pathway. Metabolomics further confirms the previous results. Collectively, these findings provide basic support for the W. coagulans as a functional food additive or a promising therapeutic agent for the effective management of UC.