Production of proinflammatory cytokines contributes to cardiac dysfunction during ischemia-reperfusion. The principal mechanism responsible for the induction of this innate stress response during periods of myocardial ischemia-reperfusion remains unknown. Toll-like receptor 2 (TLR2) is a highly conserved pattern recognition receptor that has been implicated in the innate immune response to a variety of pathogens. However, TLR2 may also mediate inflammation in response to noninfectious injury. We therefore hypothesized that TLR2 is essential for modulating myocardial inflammation and left ventricular (LV) function during ischemia-reperfusion injury. Susceptibility to myocardial ischemia-reperfusion injury following ischemia-reperfusion was determined in Langendorff-perfused hearts isolated from wild-type mice and mice deficient in TLR2 (TLR2D) and Toll interleukin receptor domain-containing adaptor protein. After ischemia-reperfusion, contractile performance was significantly impaired in hearts from wild-type mice as demonstrated by a lower recovery of LV developed pressure relative to TLR2D hearts. Creatinine kinase levels were similar in both groups after reperfusion. Contractile dysfunction in wild-type hearts was associated with elevated cardiac levels of TNF and IL-1β. Ischemia-reperfusion-induced LV dysfunction was reversed by treatment with the recombinant TNF blocking protein etanercept. These studies show for the first time that TLR2 signaling importantly contributes to the LV dysfunction that occurs following ischemia-reperfusion. Thus disruption of TLR2-mediated signaling may be helpful to induce immediate or delayed myocardial protection from ischemia-reperfusion injury.