One of the leading causes of death in the world is ischemia/reperfusion (I/R)-mediated acute myocardial infarction. There are a lot of Chinese traditional patent medicines, such as Xin'an capsules, Xin Xuening tablets, and so on, which have protective effects against myocardial I/R injury and have been routinely used in treating cardiac diseases for a long time in China. Hyperoside (Hyp) is the chief component of these medicines. This study investigated the action of Hyp in isolated myocardial I/R injury, as well as its possible mechanisms. Using the Langendorff model, isolated Sprague–Dawley rat hearts were subjected to 30 min of global ischemia and 50 min of reperfusion. Cardiac function was measured, and infarct size was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase (CK). Myocardium was also measured for total superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Phosphorylation of extracellular signal-regulated protein kinase (ERK) was analyzed by Western blotting. We report for the first time that administration of Hyp before/after I/R significantly improved heart contraction and limited the infarct size and CK and LDH leakage from the damaged myocardium after I/R. The activity of SOD and the MDA content remarkably changed in the presence of Hyp as well. Phosphorylation of ERK was significantly increased in Hyp-treated hearts compared to controls (p<0.01). Hyp-induced ERK phosphorylation was inhibited by PD98059. We therefore conclude that Hyp can protect cardiomyocytes from I/R-induced oxidative stress through the activation of ERK-dependent signaling.