The cardiotoxic adverse effects of anthracycline antibiotics limit their therapeutic utility as essential components of chemotherapy regimens for hematologic and solid malignancies. Here we show that the hematopoietic cytokine erythropoietin attenuates doxorubicin-induced apoptosis of primary neonatal rat ventricular cardiomyocytes in a dose-dependent manner. Erythropoietin treatment induced rapid, time-dependent phosphorylation of MAP kinases (MAPK) Erk1/2 and the phosphatidylinositol 3-kinase substrate Akt. Treatment of cardiomyocytes with inhibitors of phosphatidylinositol 3-kinase (LY294002) or Akt (Akti-1/2) abolished the protective effect of erythropoietin, whereas treatment with MAPK kinase (MEK1) inhibitor U0126 did not. Erythropoietin also induced the phosphorylation of GSK-3beta, a downstream target of PI3K-Akt. Because phosphorylation is known to inactivate GSK-3beta, we investigated whether GSK-3beta inhibition is cardioprotective. We found that GSK-3beta inhibitors SB216763 or lithium chloride blocked doxorubicin-induced cardiomyocyte apoptosis in a manner similar to erythropoietin, suggesting that GSK-3beta inhibition is involved in erythropoietin-mediated cardioprotection. Erythropoietin may serve as a novel cardioprotective agent against anthracycline-induced cardiotoxicity.