To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9 − / − as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis . Infected TLR9 − / − but not TLR2 − / − mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)- γ responses in vivo, but in common with TLR2 − / − animals, the TLR9 − / − mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9 − / − mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN- γ recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis , where dendritic cells and macrophages from TLR2/9 − / − mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.