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.