Background - Staphylococcus aureus sepsis is associated with significant myocardial dysfunction. Toll-like receptor 2 (TLR2) mediates the inflammatory response to S aureus and may trigger an innate immune response in the heart. We hypothesized that a TLR2 deficiency would attenuate S aureus-induced cardiac proinflammatory mediator production and the development of cardiac dysfunction. Methods and Results - Wild-type and TLR2-deficient (TLR2D) mice were studied. S aureus challenge significantly increased tumor necrosis factor, interleukin-1β, and nitric oxide expression in hearts of wild-type mice. This response was significantly blunted in TLR2D mice. Hearts from TLR2D mice had impaired S aureus-induced activation of interleukin-1 receptor-associated kinase, c-Jun NH2 terminal kinase, nuclear factor-κB, and activator protein-1. Moreover, hearts from TLR2D mice were protected against S aureus-induced contractile dysfunction. Conclusions - These results show for the first time that TLR2 signaling contributes to the loss of myocardial contractility and cytokine production in the heart during S aureus sepsis.