Objective: Postconditioning (POC), a novel strategy of cardioprotection against ischemia/reperfusion injury, is clinically attractive because of its therapeutic application at the predictable onset of reperfusion. POC activates several intracellular kinase signaling pathways, including PI3K-Akt (RISK). The regulation of POC-induced survival kinase signaling, however, has not been fully characterized. JAK-STAT activation is integral to cardiac ischemic tolerance and may provide upstream regulation of RISK. We hypothesized that postconditioning requires the activation of both JAK-STAT and RISK signaling. Methods: Langendorff perfused mouse hearts were subjected to 30 minutes global ischemia and 40 minutes reperfusion, with or without POC immediately after ischemia. A separate group of POC hearts was treated with AG490, a JAK-2 inhibitor, Stattic, a specific STAT-3 inhibitor, or LY294002 (LY), a PI3K inhibitor, at the onset of reperfusion. Cardiomyocyte-specific STAT-3 knockout (KO) hearts were also subjected to non-POC or POC protocols. Myocardial performance (+dP/dt_max, mmHg/s) was assessed throughout each perfusion protocol. Phosphorylated STAT-3 and Akt expression was analyzed by Western immunoblotting. Results: Postconditioning enhanced myocardial functional recovery and increased expression of pSTAT-3 and p-Akt. JAK-STAT inhibition abrogated POC-induced functional protection. STAT-3 inhibition decreased expression of both pSTAT-3 and p-Akt. PI3K inhibition also attenuated POC-induced cardioprotection and reduced p-Akt expression but had no effect on STAT-3 phosphorylation. Interestingly, STAT-3 KO hearts undergoing POC exhibited improved ischemic tolerance compared to KO non-POC hearts. Conclusions: Postconditioning induces myocardial functional protection by activating the RISK pathway. JAK-STAT signaling, however, is insufficient for effective postconditioning without PI3K-Akt activation.