AMP-activated protein kinase (AMPK) is a stress signaling enzyme that orchestrates the regulation of energy-generating and -consuming pathways. Intrinsic AMPK activation protects the heart against ischemic injury and apoptosis, but whether pharmacologic AMPK stimulation mitigates ischemia-reperfusion damage is unknown. The aims of this study were to determine whether direct stimulation of AMPK using a small molecule activator, A-769662, attenuates myocardial ischemia-reperfusion injury and to examine its cardioprotective mechanisms. Isolated mouse hearts pre-treated with A-769662 had better recovery of left ventricular contractile function (55% vs. 29% of baseline rate-pressure product; p=0.03) and less myocardial necrosis (56% reduction in infarct size; p<0.01) during post-ischemic reperfusion compared to control hearts. Pre-treatment with A-769662 in vivo attenuated infarct size in C57Bl/6 mice undergoing left coronary artery occlusion and reperfusion compared to vehicle (36% vs. 18%, p=0.025). Mouse hearts with genetically inactivated AMPK were not protected by A-769662, indicating the specificity of this compound. Pre-treatment with A-769662 increased the phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2), preserved energy charge during ischemia, delayed the development of ischemic contracture, and reduced myocardial apoptosis and necrosis. A-769662 also augmented endothelial nitric oxide synthase (eNOS) activation during ischemia, which partially attenuated myocardial stunning, but did not prevent necrosis. AMPK is a therapeutic target that can be stimulated by a direct-acting small molecule in order to prevent injury during ischemia-reperfusion. The use of AMPK activators may represent a novel strategy to protect the heart and other solid organs against ischemia.