Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK) which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied adult male rats, fasted overnight, using an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU/kg/min) was begun either 10 min before ischemia (InsulinBI), 5 min before reperfusion (InsulinBR) or 30 min after the onset of reperfusion (InsulinAR), and continued until the end of the study. Compared to saline control, insulin decreased the infarct size in both InsulinBI (p<0.001) and InsulinBR (p<0.03) rats, but not in Insulin_AR rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK when compared with the non-ischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg, i.v. 30 min prior to ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in Insulin_BI and Insulin_BR rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB239063, a potent and specific inhibitor of p38 MAPK, 10 min prior to reperfusion enabled insulin-mediated myocardial protection in Insulin_AR rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion and activation of p38 MAPK abolishes insulin's dioprotective effect.