The roles of innate immune responses in protection from or pathogenesis of severe leptospirosis remain unclear. We examined the role of Toll-like receptors (TLRs) in mouse infection and macrophage responses to Leptospira. C3H/HeJ mice (TLR4 deficient) and C3H/HeJ-SCID mice, but not C3H/OuJ mice (TLR4 intact), died after intraperitoneal infection with Leptospira interrogans serovar Icterohaemorrhagiae. Death in both C3H/HeJ mouse strains was associated with jaundice and pulmonary hemorrhage, similar to the patient from whom the isolate was obtained. In chronic sublethal infection, TLR4-deficient mice harbored more leptospires in liver, lung, and kidney than control mice. Heat-killed Leptospira stimulated macrophages to secrete proinflammatory cytokines, tumor necrosis factor alpha, interleukin-6, and macrophage inflammatory protein 2 not inhibited by polymyxin B, suggesting that leptospiral lipopolysaccharide (LPS) did not drive these responses. Anti-TLR4 and anti-MD-2 but not anti-CD14 monoclonal antibodies inhibited cytokine production. Peritoneal macrophages from CD14-/- and TLR2-/- mice exhibited no defect in cytokine responses to Leptospira compared to controls. Macrophages from C3H/HeJ, TLR4-/-, and MyD88-/- mice secreted far-lower levels of cytokines than wild-type macrophages in response to Leptospira. TLR4 plays a crucial role in protection from acute lethal infection and control of leptospiral burden during sublethal chronic infection. Cytokine responses in macrophages correlated with leptospiral clearance. These TLR4-dependent but CD14/TLR2-independent responses are likely mediated by a leptospiral ligand(s) other than LPS.