Gut microbiota composition of patients with ulcerative colitis (UC) is markedly altered compared with healthy individuals. There is mounting evidence that probiotic therapy alleviates disease severity in animal models and patients with inflammatory bowel disease (IBD). Bacillus subtilisis, as a probiotic, has also demonstrated a protective effect in IBD. However, the therapeutic mechanism of its action has yet to be elucidated. In the present study, a dextrose sulfate sodium (DSS)-induced UC mouse model was used to investigate the role of B. subtilis in the restoration of gut flora and determine its effective dose. Mucosal damage was assessed by performing alcian blue staining, cytokine levels were analyzed by ELISA and microbiota composition was investigated using 454 pyrosequencing to target hypervariable regions V3-V4 of the bacterial 16S ribosomal RNA gene. The results demonstrated that a higher dose B. subtilisis administration ameliorated DSS-induced dysbiosis and gut inflammation by balancing beneficial and harmful bacteria and associated anti- and pro-inflammatory agents, thereby aiding intestinal mucosa recovery from DSS-induced injuries. These findings indicate that choosing the correct dose of B. subtilis is important for effective UC therapy. The present study also helped to elucidate the mechanisms of B. subtilis action and provided preclinical data for B. subtilis use in UC therapy.