Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with α-C-GalCer, which induces a strong IFN-γ but only a weak IL-4 production by iNKT cells, in contrast to the ligands α-GalCer (both IFN-γ and IL-4) and OCH (primarily IL-4). The protective effect of α-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-γ and IL-17 production and was negated by systemic neutralization of IFN-γ. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-γ and a consequent dampening of the Th1 as well as the Th17 effector responses.