In the ischemic or hypoxic brain, astrocytes appear to be one of the main sources of erythropoietin (EPO). In this study, we investigated the differential contribution of hypoxia inducible factor (HIF) isoforms to the regulation of hypoxic EPO expression in cultured astrocytes. In addition, using an in vitro model of oxygen-glucose deprivation (OGD), we studied the role of HIF-1α and HIF-2α in the generation of paracrine protective signals by astrocytes that modulate the survival of neurons exposed to OGD. Expression of HIF-1α or HIF-2α was abrogated by infecting astrocytes with lentiviral particles encoding small interference RNA specific for HIF-1α or HIF-2α (siHIF-1α or siHIF-2α). Astrocytes infected with siHIF-1α showed abrogated hypoxic induction of vascular endothelial growth factor (VEGF) and lactate dehydrogenase (LDH) but normal EPO induction. In contrast, reduction of HIF-2α expression by siHIF-2α led to a drastic decrease of EPO hypoxic expression, but it did not affect LDH or VEGF upregulation. To further test whether HIF-2 is sufficient to drive EPO upregulation, we expressed oxygen-insensitive mutant forms of HIF-1α (mtHIF-1α) (P402A/P577A) and HIF-2α (mtHIF-2α) (P405A/P530A). Expression of mtHIF-2α but not mtHIF-1α in normoxic astrocytes resulted in a significant upregulation of EPO mRNA and protein. Accordingly, HIF-2α but not HIF-1α was found to be associated with the EPO hypoxia-response element by a chromatin immunoprecipitation assay. Interestingly, conditioned medium from astrocytes challenged by sublethal OGD improved neuronal survival to OGD; however, this effect was abolished during the downregulation of astrocytic HIF-2α using siHIF-2α. These results indicate that HIF-2α mediates the transcriptional activation of EPO expression in astrocytes, and this pathway may promote astrocytic paracrine-dependent neuronal survival during ischemia.