Abstract: To investigate the protective mechanism of bacillus licheniformis (BL) carried aboard Shenzhou-15 manned spacecraft for six months of spaceflight against dextran sulfate sodium (DSS)-induced ulcerative colitis in mice, mice were divided into a control group (CON), a DSS model group (DSS), a DSS + ground-based bacteria group (G-BL), a DSS + spaceflight bacteria group (6M-BL), and a mesalazine positive drug group (POS). Colonic H&E staining, expression detection of barrier proteins ZO-1, Occludin, and E-cadherin, and plasma metabolomic analysis were performed. The results showed that compared with the DSS group, 6M-BL significantly alleviated colonic histopathological damage, with a 58.6% decrease in pathological score, superior to the 44.8% decrease in the G-BL group. Meanwhile, the expression levels of ZO-1, Occludin, and E-cadherin were significantly upregulated by 106.8%, 133.1%, and 138.6%, respectively. Metabolomic analysis revealed that 6M-BL improved DSS-induced metabolic abnormalities, mainly involving taurine and hypotaurine metabolism, primary bile acid biosynthesis, and butyrate metabolism pathways. Compared with G-BL, 6M-BL more significantly elevated the levels of protective metabolites such as taurine, sinapic acid, and 3-(4-hydroxyphenyl) propionic acid, while reducing the levels of inflammation-related metabolites such as β-muricholic acid and xanthurenic acid. These findings suggest that 6M-BL may promote intestinal barrier repair by regulating the above pathways to synergistically inhibit inflammatory responses, thereby exerting an intervention effect on colitis.