Endothelial progenitor cells (EPCs) play an important role in both reendothelialization and neovascularization. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of ischemic diseases. However, ex vivo cultivation of EPCs leads to rapid onset of EPCs senescence, thereby severely limiting the proliferative capacity and clonal expansion potential. Therefore, we investigated whether puerarin might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic beta-galactosidase staining. Puerarin dose dependently prevented the onset of EPCs senescence in culture. Moreover, puerarin increased proliferation of EPCs as assessed by BrdU incorporation assay and colony-forming capacity. To get further insights into the underlying mechanisms of these effects induced by puerarin, we measured telomerase activity and determined the phosphorylation of serine/threonine protein kinase Akt by using western blot. Puerarin significantly increased telomerase activity and phosphorylation of Akt, a downstream effector of phosphoinositide 3-kinase (PI-3K). Moreover, pretreatment with PI-3K blockers, either wortmannin or LY294002, significantly attenuated the puerarin puerarin-induced telomerase activity. Taken together, the results of the present study indicated that puerarin delayed the onset of EPCs senescence, which may be related to the activation of telomerase through the PI-3K/Akt pathway. The inhibition of EPCs senescence by puerarin in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.