Diet has been suggested to be a potential environmental risk factor for the increasing incidence of autoimmune diseases, yet the underlying mechanisms remain elusive. Here, we show that high glucose intake exacerbated autoimmunity in mouse models of colitis and experimental autoimmune encephalomyelitis (EAE). We elucidated that high amounts of glucose specifically promoted T helper-17 (Th17) cell differentiation by activating transforming growth factor- (TGF-) from its latent form through upregulation of reactive oxygen species (ROS) in T cells. We further determined that mitochondrial ROS (mtROS) are key for high glucose-induced TGF- activation and Th17 cell generation. We have thus revealed a previously unrecognized mechanism underlying the adverse effects of high glucose intake in the pathogenesis of autoimmunity and inflammation.