Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis. •Dietary fatty acids have profound influence on T cell differentiation in the gut•Middle- and long-chain fatty acids (LCFAs) support Th1 and Th17 cell differentiation•Short-chain fatty acids (SCFAs) lead to increased Treg cell differentiation•LCFAs worsen disease in an animal model of MS; SCFAs exert the opposite effect Haghikia and colleagues show that dietary fatty acids (FAs) influence T cell differentiation in the gut, with short FAs leading to increased Treg cell differentiation and long FAs supporting Th1 and/or Th17 cell differentiation. These FAs differentially affect EAE severity, demonstrating a direct dietary impact on central nervous system autoimmunity.