Objective To investigate the effects of shear stress on late endothelial progenitor cells (EPCs) functions in vitro and in vivo. Methods Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in EGM-2MV and induced into EPCs. The 3rd~4th generation of EPCs, namely late EPCs, were treated with shear stress (1.2 Pa). Then cell biological functions, such as proliferation, adhesion, migration and ability of tube formation, were assayed with EdU incorporation assay, adhesion testing, Boyden chamber assay and Matrigel, respectively. The gene expression of VEFG was analyzed by real time RT-PCR. The apoptosis and aging situation of late EPCs were assayed by FACS and senescence-associated β-galactosidase (SA-β-gal) staining. The reendothelialization capacity of late EPCs treated by shear stress was evaluated by establishing models of freshly balloon-injured carotid arteries of rats and cell transplantation in situ. Results Shear stress could increase proliferation, adhesion, migration and tube formation of late EPCs (P＜0.05), upregulate the gene expression of VEGF, inhibit EPC apoptosis and delayed EPC aging (P＜0.05). Transplantation of late EPCs treated by shear stress facilitated in vivo reendothelialization in the injured arterial segment and inhibited neointima formation. Conclusions Shear stress within the physiological range can improve the functions of late EPCs and enhance their therapeutic ability of repairing vascular endothelial injury, which provides experimental basis for the clinic application of EPCs and shear stress-mediated cell therapy.