Natural killer dendritic cells (NKDC) are a novel subtype of dendritic cells with natural killer (NK) cell properties. IFN-γ is a pleiotropic cytokine that plays an important role in the innate immune response to tumors. Based on our previous finding that the combination of Toll-like receptor 9 ligand CpG and interleukin (IL)-4 stimulates NKDC to produce IFN-γ, we hypothesized that NKDC are the major IFN-γ-producing dendritic cell subtype and may play a significant role in the host antitumor response. We found that under several conditions in vitro and in vivo NKDC accounted for the majority of IFN-γ production by murine spleen CD11c⁺ cells. IL-18 alone induced NKDC to secrete IFN-γ, and the combination of IL-18 and CpG resulted in a synergistic increase in IFN-γ production, both in vitro and in vivo. NK cells made 26-fold less IFN-γ under the same conditions in vitro, whereas dendritic cells produced a negligible amount. The mechanism of IFN-γ secretion by NKDC depended on IL-12. NKDC selectively proliferated in vitro and in vivo in response to the combination of IL-18 and CpG. Systemic treatment with IL-18 and CpG reduced the number of B16F10 melanoma lung metastases. The mechanism depended on NK1.1⁺ cells, as their depletion abrogated the effect. IL-18 and CpG activated NKDC provided greater tumor protection than NK cells in IFN-γ^−/− mice. Thus, NKDC are the major dendritic cell subtype to produce IFN-γ. The combined use of IL-18 and CpG is a viable strategy to potentiate the antitumor function of NKDC. (Cancer Res 2006; 66(21): 10497-504)