The high rate of mortality in patients with sepsis results from an inappropriately amplified systemic inflammatory response to infection. Toll-like receptors (TLRs) are important for the activation of innate immunity against microbial pathogens. We demonstrate a critical role of TLR9 in the dysregulated immune response and death associated with sepsis. Compared with wild-type (WT) mice, TLR9^−/− mice exhibited lower serum inflammatory cytokine levels, higher bacterial clearance, and greater survival after experimental peritonitis induced by cecal ligation and puncture (CLP). Protection of TLR9^−/− mice after CLP was associated with a greater number of peritoneal dendritic cells (DCs) and granulocytes than in WT controls. Adoptive transfer of TLR9^−/− DCs was sufficient to protect WT mice from CLP and increased the influx of peritoneal granulocytes. Subsequent experiments with a depleting antibody revealed that granulocytes were required for survival in TLR9^−/− mice. Remarkably, a single injection of an inhibitory CpG sequence that blocks TLR9 protected WT mice, even when administered as late as 12 h after CLP. Our findings demonstrate that the detrimental immune response to bacterial sepsis occurs via TLR9 stimulation. TLR9 blockade is a potential strategy for the treatment of human sepsis.