Ischemic preconditioning (IPC) is the most powerful endogenous cardioprotective form of cellular adaptation. However, the inhibitory or augmenting mechanism underlying cardioprotection via IPC remains largely unknown. Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible potent negative feedback regulator of the signal transducer and activator of transcription-3 (STAT3) signaling pathway. Here, we aimed to determine whether cardiac SOCS3 deficiency and IPC would synergistically reduce infarct size after myocardial ischemia reperfusion injury. We evaluated STAT3 activation and SOCS3 induction after ischemic conditioning (IC) using western blot analysis and real-time PCR, and found that myocardial IC alone transiently activated myocardial STAT3 and correspondingly induced SOCS3 expression in wild-type mice. Compared with wild-type mice, cardiac-specific SOCS3 knockout (SOCS3-CKO) mice showed significantly greater and more sustained IC-induced STAT3 activation. Following ischemia reperfusion, IPC substantially reduced myocardial infarct size and significantly enhanced STAT3 phosphorylation in SOCS3-CKO mice compared to in wild-type mice. Real-time PCR array analysis revealed that SOCS3-CKO mice after IC exhibited significantly increased expressions of several anti-apoptotic genes and SAFE pathway-related genes. Moreover, real-time PCR analysis revealed that myocardial IC alone rapidly induced expression of the STAT3-activating cytokine erythropoietin in the kidney at 1 h post-IC. We also found that the circulating erythropoietin level was promptly increased at 1 h after myocardial IC. Myocardial SOCS3 deficiency and IPC exert synergistic effects in the prevention of myocardial injury after ischemia reperfusion. Our present results suggest that myocardial SOCS3 is a potent inhibitor of IPC-induced cardioprotection, and that myocardial SOCS3 inhibition augment IPC-mediated cardioprotection during ischemia reperfusion injury.