Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia/reperfusion injury and that cytokines link TLR4 to post-ischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia/reperfusion is unknown. In the present study we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia/reperfusion, 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function, and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia/reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery following ischemia/reperfusion. Recombinant HSC70 induced NF-B activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore this study demonstrates for the first time that: 1) the myocardium releases HSC70 during ischemia/reperfusion, 2) extracellular HSC70 contributes to the post-ischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function following ischemia/reperfusion.