In vivo administration of NKT cell ligand, α-galactosylceramide (α-GalCer), caused the activation of NKT cells to induce a strong NK activity and cytokine production by CD1d-restricted mechanisms. Surprisingly, we also found that α-GalCer induced the activation of immunoregulatory cells involved in acquired immunity. Specifically, in vivo administration of α-GalCer resulted in the induction of the early activation marker CD69 on CD4⁺ T cells, CD8⁺ T cells and B cells in addition to macrophages and NKT cells. However, no significant induction of CD69 was observed on cells from CD1d- or V_α14 NKT-deficient mice, indicating an essential role for the interaction between NKT cells and CD1d-expressing dendritic cells (DC) in the activation of acquired immunity in response to α-GalCer. Indeed, in vivo injection of α-GalCer resulted not only in the activation of NKT cells but also in the generation of CD69⁺CD8⁺ T cells possessing both cytotoxic T lymphocyte (CTL) activity and IFN-γ-producing ability. Tumor-specific CTL generation was also accelerated by α-GalCer. The critical role of CD40–CD40 ligand (CD40L)-mediated NKT–DC interaction during the development of CD69⁺CD8⁺ CTL by α-GalCer was demonstrated by blocking experiments using anti-CD40L mAb. These findings provide direct evidence for a critical role of CD1d-restricted NKT cells and DC in bridging innate and acquired immunity.