In its vertebrate host, Leishmania encounters cells that express TLRs. Using genetically resistant C57BL/6 mice deficient in either TLR2, 4, or 9, we show in this study that only TLR9-deficient mice are more susceptible to infection with Leishmania major. TLR9-deficient mice resolved their lesions and controlled parasites growth with much lower efficiency than wild-type C57BL/6 mice. The absence of TLR9 also transiently inhibited the development of curative Th1 response. In an attempt to analyze the possible basis for such aberrant response in TLR9(-/-) mice, we have studied the importance of TLR9 for the activation of dendritic cells (DCs) by L. major. Results show that DCs in the draining lymph nodes are activated following infection with L. major. Furthermore, bone marrow-derived DCs as well as DCs freshly isolated from the spleen of C57BL/6 mice can be activated by either heat-killed or live L. major in vitro. In sharp contrast, L. major failed to activate DCs from TLR9(-/-) mice. Noteworthily, activation of DCs was abolished either following treatment of the parasites with DNase or after acidification of the endosomal compartment of DCs by chloroquine, pinpointing the DNA of L. major as the possible ligand of TLR9 leading to the activation of DCs. Results showed that DNA purified from L. major was indeed capable of activating DCs in a strictly TLR9-dependent manner. Moreover we showed that the L. major DNA-induced TLR9 signaling in DCs condition these cells to promote IFN-gamma production by CD4(+) T cells.