Natural killer T cells (NKT cells) expressing a semi-invariant CD1d-reactive T cell receptor (invariant NKT, iNKT) can be rapidly activated by monocytes or immature dendritic cells (iDCs) bearing a CD1d-presented glycolipid antigen and can in turn stimulate these myeloid cells to mature and produce IL-12. Previous studies have shown that iNKT-produced IFNgamma and CD40 ligand contribute to this dendritic cell maturation. This study demonstrates that CD1d ligation alone, in the absence of iNKT, could rapidly (within 24 h) stimulate production of bioactive IL-12p70 by CD1d+ human peripheral blood monocytes as well as iDCs. IFNgamma alone had no effect, but it markedly enhanced CD1d-stimulated IL-12 production. Monocyte differentiation, as assessed by CD40 and CD1a up-regulation, was also accelerated by CD1d stimulation, consistent with this representing a physiological response. CD1d ligation on the human monocytic cell line THP-1 similarly specifically stimulated IL-12 production. Biochemical studies showed that IL-12 release correlated with rapid phosphorylation of IkappaB, a critical step in NF-kappaB activation. Selective NF-kappaB inhibition blocked this CD1d-stimulated IL-12 production. Finally, CD1d ligation could also enhance IL-12 production in the presence of suboptimal LPS or CD40 stimulation. These findings demonstrate an innate immune signaling function for CD1d and provide a mechanism for the rapid activation of monocytes and iDCs by CD1d-reactive T cells.