To investigate the influence of HIF-1alpha overexpression on the differentiation of endothelial progenitor cells (EPCs) ex vivo, EPCs were isolated from human peripheral blood by density gradient centrifugation, overexpressed HIF-1alpha was transfected to EPCs by electroporation; HIF1alpha, HIF1beta, vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1alpha protein was detected with immunohistochemistry in a time course. CD31(+) cells were measured with flow cytometry. Cell morphology was observed after transfection. The results showed that the transfection efficiency of HIF-1alpha to EPCs was about 20%. HIF-1alpha and its controlled target gene VEGF were markedly induced by HIF-1alpha vector (P < 0.05). HIF1beta had its same level as it before interference (P > 0.05). HIF-1alpha protein was induced by HIF-1alpha transfection after 12 hours but was undetectable at 24 hours. After 7 - 14 days cultured in 21% oxygen pressure, fluorescence-trace experiments revealed that CD31 + EPCs/EC could be generated more efficiently from overexpressed HIF-1alpha than that from pEGFP transfected group (P > 0.05). EPC morphology was observed by light microscopy. HIF-1alpha-transfected cells under normoxia sprouted more rapidly from the EPC colonies than the untransfected cells or cells transfected with an GFP vector, which essentially maintained the original colony formation. HIF-1alpha transfected cells took on an array-like arrangement rather than random dispersal, suggesting that they were in an advanced state of differentiation. It is concluded that the utility of overexpression of HIF-1alpha can induce target genes which have influence on cell differentiation. HIF-1alpha transfection was found to give a prospected way to do the insight research on ischemic treatment in vivo.