AbstractInvariant NKT (inv. NKT) cells co‐express an invariant α β T cell receptor and the NK receptor NK1.1 and, upon CD1d‐restricted recognition of the glycosphingolipid antigen α‐galactosyl ceramide (αGalCer), secrete large amounts of regulatory cytokines. We investigated whether αGalCer‐dependent activation of inv. NKT cells protects from experimental autoimmune encephalomyelitis (EAE), an immune‐mediated disease of the central nervous system mimicking multiple sclerosis, induced in C57BL/6 mice by the myelin oligodendrocyte glycoprotein (MOG) encephalitogenic peptide aa 35–55. αGalCer was administered at the time of immunization s.c., mixed with complete Freund's adjuvant and MOG35‐55 peptide, or administered i.p., diluted in PBS. EAE onset was delayed and disease severity was decreased only when αGalCer was s.c. administered. The protective effect of s.c. administration of αGalCer was associated with a markedly enhanced IFN‐γ production by liver‐confined inv. NKT cells which, in turn, suppressed Th1‐cytokine production and fostered secretion of IL‐10 from MOG35–55‐specific T cells. In vivo neutralization of IFN‐γ, but notIL‐4, reversed the protective effect induced by s.c. administration of αGalCer, further confirming the critical regulatory role exerted by IFN‐γ‐producing inv. NKT cells. Our results indicate that αGalCer, properly administered, may elicit an inv. NKT‐cell‐mediated suppressive effect on the effector function of encephalitogenic T cells; this effect is able to ameliorate autoimmunedemyelination.