There is a close association between hyperglycemia and increased risk of mortality after acute myocardial infarction (AMI). However, whether acute hyperglycemia exacerbates myocardial ischemia/reperfusion (MI/R) injury remains unclear. We observed the effects of acute hyperglycemia on MI/R injury and on the cardioprotective effect of glucose-insulin-potassium (GIK). Male rats were subjected to 30 minutes of myocardial ischemia and 6 hours of reperfusion. Rats were randomly received one of the following treatments (i.v. infusion at 4 ml·kg^-1·h^-1): Vehicle, GIK (GIK during reperfusion; glucose: 200g/L, insulin: 60 U/L, KCL: 60 mmol/L), HG (high glucose during ischemia; glucose:500 g/L), GIK plus HG (HG during I and GIK during R) or GIK plus wortmannin (GIK during R and wortmannin 15 mins before R). Blood glucose and left ventricular pressure (LVP) were monitored throughout the experiments. Hyperglycemia during ischemia not only significantly increased myocardial apoptosis (23.6±1.7% vs. 18.8±1.4%, P<0.05 vs. vehicle), increased infarct size (IS) (45.6±3.0% vs. 37.6±2.0%, P<0.05 vs. vehicle), decreased Akt and GSK-3 phosphorylations (1.7±0.2% and 2.3±0.2% fold of vehicle, respectively, P<0.05 vs. vehicle) following MI/R, but almost completely blocked the cardioprotective effect afforded by GIK, as evidenced by significantly increased apoptotic index (19.1±2.0% vs. 10.3±1.2%, P<0.01 vs. GIK), increased myocardial IS (39.2±2.8% vs. 27.2±2.1%, P<0.01 vs. GIK), decreased Akt phosphorylation (1.1±0.1% vs. 1.7±0.2%, P<0.01 vs. GIK) and GSK-3 phosphorylation (1.4±0.2% vs. 2.3±0.2%, P<0.05 vs. GIK). Hyperglycemia significantly exacerbates MI/R injury and blocks the cardioprotective effect afforded by GIK, which is, at least in part, due to hyperglycemia-induced decrease of myocardial Akt activation.