Although the etiology of Alzheimer's disease (AD) is not fully understood, multiple lines of evidence suggests the importance of amyloid-beta (Abeta) in the initiation/progression of the disease. In this study, we investigated protective effects of erythropoietin (EPO) on Abeta(25-35)-induced cell death in cultured rat pheochromocytoma cells (PC12 cells). EPO (2U/ml) in combination with Abeta(25-35) increased the cell viability and reduced the number of apoptotic cells by MTT assay, Trypan blue dye exclusion method, TUNEL staining and Hoechst 33342 staining. In mechanistic study, EPO induced time-dependent phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt. Treatment of PC12 cells with PI3K inhibitors LY294002 abolished the protective effects of EPO. EPO also induced the phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), a downstream target of PI3K/Akt, and GSK-3beta inhibitors lithium chloride blocked Abeta(25-35)-induced cell apoptosis in a manner similar to EPO, suggesting that GSK-3beta inhibition is involved in EPO-mediated cytoprotection. Moreover, the expression of anti-apoptotic protein Bcl-2 was increased by EPO involving PI3K/Akt pathway. These studies demonstrate that EPO is an effective neuroprotective agent and is a viable candidate for treating AD.