Aim To test the hypothesis that exogenous administration of HIF1α could enhance the differentiation of endothelial progenitor cells (EPC) in hypoxia in vitro. Methods EPC were isolated from human peripheral blood by density gradient centrifugation. We transfected overexpressed HIF1αto EPC in normoxia/hypoxia by electroporation and measured the transfection effiency. HIF1α, HIF1β(ARNT), vascular endothelial growth factor (VEGF), nitric oxide (NO) level were measured with RT-PCR, Western blot, flow cytometry or Elisa. EPC morphology were also observed. Results Approximately 20% pEGFP+cells were observed after 36 h transfection. Compared with normoxia,the expression of both HIF1α mRNA and ARNT mRNA was not at all augmented in response to hypoxic stimulus (p<0.05). In contrast,VEGF mRNA expression was significantly up-regulated under hypoxia(p<0.05). HIF1α western bolt showed hypoxia stabilization of HIF1α protein was induced at 3 h, 6 h and 12 h in hypoxia while the expression of HIF1α protein was undetectable at 24 h in hypoxia. After 6-13 days cultured in 21% or 1% oxygen pressure, fluorescence-trace experiments revealed that CD31+EPC could be generated more effienciently from overexpressed HIF1α in hypoxia than that of pEGFP transfected EPC (p<0.05) (53.8%±3.7% vs 40.2%±4.3% under normoxia at d6; 66.2%±6.5% vs 51.8%±3.5% under normoxia at d13; 60.2%±5.0% vs 46.8%±3.5% under hypoxia at d6; 76.1%±1.9% vs 59.0%±3.5% under hypoxia at d13). A time kinetic NO was measured by Elisa Kit which was markedly enhanced by HIF1α in hypoxia (p<0.01). EPC differentiation was also observed by cell morphology. EPC differentiation and proliferation was more rapid in overexpressing of HIF1α group in hypoxia than in normoxia. Conclusions Hypoxia is ideal for EPC differentiation and proliferation. Overexpressing of HIF1α is important for exploring HIF1-dependent processes and for hypoxia-induced pathophysiological events. Moreover, HIF1α transfection was found to give a prospected way to do the insight research on ischemic treatment in vivo.