CD1d-reactive natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and/or Th2 cytokines, can activate antigen-presenting cell (APC) interleukin-12 (IL-12) production, and are implicated in the regulation of adaptive immune responses. The role of the CD1d system was assessed during infection with encephalomyocarditis virus (EMCV-D), a picornavirus that causes acute diabetes, paralysis and myocarditis. EMCV-D resistance depends on IL-12-mediated interferon-γ (IFN-γ) production. CD1d-deficient mice, which also lack CD1d-reactive NKT cells, were substantially more sensitive to infection with EMCV-D. Infected CD1d knockout mice had decreased IL-12 levels in vitro and in vivo, and indeed were protected by treatment with exogenous IL-12. IFN-γ production in CD1d knockout mice was decreased compared with that in wild-type (WT) mice in response to EMCV-D in vitro, although differences were not detected in vivo. Treatment with anti-asialo-GM1 antibody, to deplete NK cells, caused a marked increase in susceptibility of WT mice to EMCV-D infection, whereas CD1d knockout mice were little affected, suggesting that NK-cell-mediated protection is CD1d-dependent. Therefore, these data indicate that CD1d is essential for optimal responses to acute picornaviral infection. We propose that CD1d-reactive T cells respond to early immune signals and function in the innate immune response to a physiological viral infection by rapidly augmenting APC IL-12 production and activating NK cells.