Exercise increases serum opioid levels and improves cardiovascular health. Here, we tested the hypothesis that opioids contribute to the acute cardioprotective effects of exercise using a rat model of exercise induced cardioprotection. For the standard protocol, rats were randomized to 4 days of treadmill training and one day of vigorous exercise (day 5), or to a sham exercise control group. On day 6, animals were sacrificed, and global myocardial ischemic tolerance was assessed on a modified Langendorff apparatus. Twenty minutes of ischemia followed by three hours of reperfusion resulted in a mean infarct size of 42% ± 4% in hearts from sham exercise controls and 21% ± 3% (p < 0.001) in the exercised group. The cardioprotective effects of exercise were gone by five days after the final exercise period. To determine the role of opioid receptors in exercise-induced cardioprotection, rats were exercised according to the standard protocol; however, just prior to exercise on days 4 and 5, rats were injected subcutaneously with 10 mg/kg of the opioid receptor antagonist naltrexone. Similar injections were performed in the sham exercise control group. Naltrexone had no significant effect on baseline myocardial ischemic tolerance in controls (infarct size 43% ± 4%). In contrast, naltrexone treatment completely blocked the cardioprotective effect of exercise (infarct size 40% ± 5%). Exercise was also associated with an early increase in myocardial mRNA levels for several opioid system genes, and with sustained changes in a number of genes that regulate inflammation and apoptosis. These findings demonstrate that the acute cardioprotective effects of exercise are mediated, at least in part, through opioid receptor-dependent mechanisms that may include changes in gene expression.