In this study, experiments were performed to determine the contribution of TLR9 to the generation of protective innate immunity against virulent bacterial pathogens of the lung. In initial studies, we found that the intratracheal administration of Klebsiella pneumoniae in wild-type (WT) BALB/c mice resulted in the rapid accumulation of dendritic cells (DC) expressing TLR9. As compared with WT mice, animals deficient in TLR9 (TLR9−/−) displayed significantly increased mortality that was associated with a> 50-fold increase in lung CFU and a> 400-fold increase in K. pneumoniae CFU in blood and spleen, respectively. Intrapulmonary bacterial challenge in TLR9−/− mice resulted in reduced lung DC accumulation and maturation as well as impaired activation of lung macrophages, NK cells, and αβ and γδ T cells. Mice deficient in TLR9 failed to generate an effective Th1 cytokine response following bacterial administration. The adoptive transfer of bone marrow-derived DC from syngeneic WT but not TLR9−/− mice administered intratracheally reconstituted antibacterial immunity in TLR9−/− mice. Collectively, our findings indicate that TLR9 is required for effective innate immune responses against Gram-negative bacterial pathogens and that approaches to maximize TLR9-mediated DC responses may serve as a means to augment antibacterial immunity in pneumonia.