This study explored the effect of plant-derived indigo supplementation on intestinal inflammation using in vivo, in vitro, and clinical sample analyses. Our results showed that indigo decreased mucosal inflammation by regulating CD4(+) T cell differentiation in a gut microbiota-dependent manner. Microbes transferred from indigo-treated mice, indigo-induced enrichment of Roseburia intestinalis, and its metabolite butyrate played a role in Th17/Treg immunity similar to that of indigo in intestinal inflammation, which was involved in mTORC1/HIF-1alpha signal-mediated reprogrammed glucose metabolism. We further showed that patients with ulcerative colitis exhibited significant gut dysbiosis and CD4(+) T cell differentiation abnormalities. Our findings provide new insights into the gut-immune axis in ulcerative colitis, offering a novel microbial-based immunotherapy for the treatment of inflammatory bowel disease.