Glycolipid-reactive Valpha24(+) invariant natural killer T (iNKT) cells have been implicated in regulating a variety of immune responses and in the induction of immunologic tolerance. Activation of iNKT cells requires interaction with professional antigen-presenting cells, such as dendritic cells (DCs). We have investigated the capacity of distinct DC subsets to modulate iNKT cell functions. We demonstrate that tolerogenic DCs (tolDCs), generated by treatment of monocyte-derived DC with interleukin (IL)-10, induced regulatory functions in human iNKT cells. tolDCs, compared with immunogenic DCs, had reduced capacity to induce iNKT-cell proliferation, but these cells produced large amounts of IL-10 and acquired an anergic phenotype. These anergic Valpha24(+) iNKT cells were able to potently inhibit allogeneic CD4(+) T-cell proliferation in vitro. Furthermore, the anergic Valpha24(+) iNKT cells could suppress DC maturation in vitro. We conclude that the interaction of iNKT cells with tolDCs plays an important role in the immune regulatory network, which might be exploited for therapeutic purposes.