Autoimmunity is a complex trait disease where the environment influences susceptibility to disease by unclear mechanisms. T cell receptor clustering and signaling at the immune synapse, T cell proliferation, CTLA-4 endocytosis, TH1 differentiation, and autoimmunity are negatively regulated by β1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. β1,6GlcNAc-branched N-glycan expression in T cells is dependent on metabolite supply to UDP-GlcNAc biosynthesis (hexosamine pathway) and in turn to Golgi N-acetylglucosaminyltransferases Mgat1, -2, -4, and -5. In Jurkat T cells, β1,6Glc-NAc-branching in N-glycans is stimulated by metabolites supplying the hexosamine pathway including glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not by control metabolites mannosamine, galactose, mannose, succinate, or pyruvate. Hexosamine supplementation in vitro and in vivo also increases β1,6GlcNAc-branched N-glycans in naïve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, TH1 differentiation, experimental autoimmune encephalomyelitis, and autoimmune diabetes in non-obese diabetic mice. Our results indicate that metabolite flux through the hexosamine and N-glycan pathways conditionally regulates autoimmunity by modulating multiple T cell functionalities downstream of β1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.