This study was designed to examine whether physiologically tolerable insulin, which maintains lower blood glucose, can protect the myocardium against ischemia/reperfusion (I/R) injury in a preclinical large animal model. Adult dogs were subjected to 50 minutes of myocardial ischemia (80% reduction in coronary blood flow) followed by 4 hours of reperfusion and treated with vehicle, glucose-insulin-potassium (GIK; glucose, 250 g/L; insulin, 60 U/L; potassium, 80 mmol/L), GK, or low-dose insulin (30 U/L) 10 minutes before reperfusion. Treatment with GIK exerted significant cardioprotective effects as evidenced by improved cardiac function, improved coronary blood flow, reduced infarct size, and myocardial apoptosis. In contrast, treatment with GK increased blood glucose level and aggravated myocardial I/R injury. It is interesting that treatment with insulin alone at the dose that reduced blood glucose to a clinically tolerable level exerted significant cardioprotective effects that were comparable to that seen in the GIK-treated group. This low-dose insulin had no effect on coronary blood flow after reperfusion but markedly reduced coronary reactive hyperemia and switched myocardial substrate uptake from fat to carbohydrate. Our results suggest that lower glucose supply to the ischemic myocardium at early reperfusion may create a "metabolic postconditioning" and thus reduce myocardial ischemia/reperfusion injury after prolonged reperfusion.