Invariant natural killer T (iNKT) cells are a subset of innate lymphocytes that recognize lipid antigens in the context of CD1d and mediate potent immune regulatory functions via the rapid production of interferon-γ (IFN-γ) and interleukin-4 (IL-4). We investigated whether diverse Toll-like receptor (TLR) signals in myeloid dendritic cells (DCs) could differentially stimulate iNKT cells. Together with the lipopolysaccharide-detecting receptor TLR4, activation of the nucleic acid sensors TLR7 and TLR9 in DCs were particularly potent in stimulating iNKT cells to produce IFN-γ, but not IL-4. iNKT cell activation in response to TLR9 stimulation required combined synthesis of type I interferon and de novo production of charged β-linked glycosphingolipid(s) by DCs. In addition, DCs stimulated via TLR9 activated both iNKT cells and NK cells in vivo and protected mice against B16F10-induced melanoma metastases. These data underline the role of TLR9 in iNKT cell activation and might have relevance to infectious diseases and cancer.