Inflammatory bowel disease (IBD) is a chronic immune-related disease, which can occur through the dysfunction of the immune system caused by the imbalance of gut microbiota. Previous studies have reported the beneficial effects of Bifidobacterium on colitis, while the related mechanisms behind these effects have not been fully elucidated. The aim of our study is to investigate the alleviation effect of Bifidobacterium animalis subsp. lactis XLTG11 (B. lactis) on dextran sulfate sodium (DSS)-induced colitis and its potential mechanism. The results showed that B. lactis XLTG11 significantly decreased weight loss, disease activity index score, colon shortening, myeloperoxide activity, spleen weight, and colon tissue damage. Additionally, B. lactis XLTG11 significantly decreased the levels of pro-inflammatory cytokines and increased the level of anti-inflammatory cytokine. Meanwhile, high doses of B. lactis XLTG11 significantly up-regulated the expression of tight junction proteins and inhibited activation of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MYD88)/nuclear factor-κB (NF-κB) signaling pathway. Furthermore, B. lactis XLTG11 increased the gut microbiota diversity and modulated gut microbiota composition caused by DSS. Moreover, Spearman’s correlation analysis also found that several specific gut microbiota were significantly correlated with colitis-related indicators. These results demonstrated that B. lactis XLTG11 can alleviate DSS-induced colitis by inhibiting the activation of the TLR4/MYD88/NF-κB signaling pathway, regulating inflammatory cytokines, improving intestinal barrier function, and modulating the gut microbiota.