Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic model for T cell-mediated autoimmunity. V(alpha)14 natural killer T (NKT) cells are a subset of T lymphocytes that recognize glycolipid antigens presented by the nonpolymorphic major histocompatibility complex (MHC) class I-like protein CD1d. Here, we show that activation of V(alpha)14 NKT cells by the glycosphingolipid alpha-galactosylceramide (alpha-GalCer) protects susceptible mice against EAE. beta-GalCer, which binds CD1d but is not recognized by NKT cells, failed to protect mice against EAE. Furthermore, alpha-GalCer was unable to protect CD1d knockout (KO) mice against EAE, indicating the requirement for an intact CD1d antigen presentation pathway. Protection of disease conferred by alpha-GalCer correlated with its ability to suppress myelin antigen-specific Th1 responses and/or to promote myelin antigen-specific Th2 cell responses. alpha-GalCer was unable to protect IL-4 KO and IL-10 KO mice against EAE, indicating a critical role for both of these cytokines. Because recognition of alpha-GalCer by NKT cells is phylogenetically conserved, our findings have identified NKT cells as novel target cells for treatment of inflammatory diseases of the central nervous system.