Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Herein, we examined any protective effects of the long-acting erythropoietin (EPO) analogue Darbepoetin alpha (DA) in a rat model of ischemia/reperfusion (I/R) injury. Rats were subjected to 30 min of ischemia followed by 72 hrs of reperfusion. In a dose response study, DA (2, 7, 11, and 30 µg/kg) or Vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high-dose of DA (30 µg/kg) was given either at the initiation or end of ischemia, or at 1 or 24 hrs after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8±3.5% (Vehicle) to 11.0±3.3% in a dose-dependent manner while there was no difference in the ischemic area between groups. Treatment with DA as late as 24 hrs after the beginning of the reperfusion still demonstrated a significant reduction in infarct size (17.0±1.6 %). Consistent with infarction data, DA improved in vivo cardiac reserve compared to Vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function apparently through a reduction of myocyte apoptosis. Of clinical importance pointing towards a relevant therapeutic utility, we report that even if given 24 hrs after I/R injury, DA can significantly protect the myocardium.