The gut-brain axis is an emerging factor in promoting multiple sclerosis (MS). However, the underlying mechanisms and strategies to target this axis are not established. Here, we investigated how the gut environment influences myelin-specific Th17 cell pathogenicity. We used the adoptive Th17 cell transfer experimental autoimmune encephalomyelitis (EAE) model and antibiotic treatment to disrupt the intestinal microbiome. We observed reduced pathogenic Th17 cell signature in the colon of antibiotic-treated mice. Treatment with fecal filtrates enhanced myelin-specific Th17 cell encephalitogenic properties both in vitro and in vivo. Fecal metabolomic profiling identified altered tryptophan-derived metabolites, including indole-3-carboxylate (I3CA). Oral I3CA supplementation accelerated EAE development. I3CA concentration in blood samples from persons with MS (PwMS) was associated with increased disease severity mirrored by increased serum neurofilament light chain levels. Altogether, our study shows that microbiota-derived metabolites play a key role in the intestine during neuroinflammation, offering potential therapeutic insights for PwMS.