Objectives: Remifentanil preconditioning attenuates myocardial ischemia reperfusion injury, but the underlying mechanism is incompletely understood. The Janus activated kinase-2 (JAK2)/signal transducers and activators of transcription-3 (STAT3) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways are critical in both ischemic and pharmacologic preconditioning cardioprotection, which involve the inactivation of glycogen synthase kinase-3 beta. We hypothesized that remifentanil preconditioning confers cardioprotection via the JAK2/STAT3 and/or PI3K/Akt activation-mediated glycogen synthase kinase-3 beta inhibition. Design: Pharmacologic intervention. Setting: Research laboratory. Subjects: Male Sprague-Dawley rats. Interventions: In vivo and in vitro treatments. Measurements and Main Results: Male Sprague-Dawley rats (n = 6 per group) were sham operated or subjected to myocardial ischemia reperfusion injury. The JAK2 inhibitor AG490 (3 mg/kg), the PI3K inhibitor wortmannin (15 mu g/kg), or the glycogen synthase kinase-3 beta inhibitor SB216763 (600 mu g/kg) were given before inducing in vivo myocardial ischemia reperfusion injury achieved by occluding coronary artery for 30 minutes followed by 120 minutes of reperfusion in the absence or presence of remifentanil preconditioning (6 mu g/kg/min). Also, isolated rat hearts were Langendorff perfused and subjected to 30 minutes of global ischemia and 120 minutes of reperfusion without or with remifentanil preconditioning (100 ng/mL) in the presence or absence of AG490 and/or SB216763. Isolated rat cardiomyocytes and H9C2 cells were subjected to hypoxia/reoxygenation alone or in combination with AG490 (100 mu M), wortmannin (100 nM), or SB216763 (3 mu M) without or with remifentanil preconditioning (2.5 mu M). Remifentanil preconditioning reduced postischemic myocardial infarction and hemodynamic dysfunction induced by myocardial ischemia reperfusion injury concomitant with increased phosphorylation of STAT3 at tyr-705 (p-STAT3) and glycogen synthase kinase-3 beta but not Akt. AG490 but not wortmannin cancelled remifentanil preconditioning cardioprotection, and SB216763 restored it despite the presence of AG490. In Langendorff-perfused hearts, AG490-mediated cancellation of remifentanil preconditioning cardioprotection in attenuating postischemic myocardial infarction and creatinine kinase-MB release was reverted by concomitant administration of SB216763. Remifentanil preconditioning also attenuated posthypoxic cardiomyocyte injury and increased p-STAT3 and glycogen synthase kinase-3 beta in isolated primary cardiomyocytes and H9C2 cells. STAT3 gene knockdown with specific synthetic RNA cancelled remifentanil preconditioning cardioprotection, whereas glycogen synthase kinase-3 beta gene knockdown, which per se did not affect STAT3 under hypoxia/reoxygenation condition, preserved remifentanil preconditioning cardioprotection regardless of STAT3 abrogation. Conclusions: Remifentanil preconditioning confers cardioprotection primarily via activation of JAK2/STAT3 signaling that can function independent of PI3K/Akt activation. Glycogen synthase kinase-3 beta is a critical downstream effector of remifentanil preconditioning cardioprotection.