Objective To investigate the effects of shear stress and vascular smooth muscle cells (VSMCs) on the proliferation of endothelial cells (ECs) and the molecular mechanism involved in. Method Using parallel-plate flow chamber system, normal shear stress of 15 dyn/cm2(1 dyn=10-5 N) was applied to ECs cultured singly and cocultured with VSMCs respectively. Then, the expression of PCNA, a molecule representing cell proliferation ability, and phosphorylation of Akt were analyzed by Western blotting in order to investigate the roles of shear stress and VSMCs in EC proliferation. Under the static condition, the expressions of PCNA and p-Akt were analyzed in ECs co-cultured with VSMCs with and without physical contact. And then TGFβ1 neutralizing antibody was employed to demonstrate the contribution of TGFβ1 in VSMCs induced EC proliferation. Results Normal shear stress decreased EC proliferation and Akt phosphorylation. VSMCs increased EC proliferation and Akt phosphorylation in both co-culture conditions with and without physical contact. Normal shear stress partly reversed the increase of proliferation and Akt phosphorylation in ECs with physical contact to VSMCs, and TGFβ1 neutralizing antibody exerted the similar effects in ECs without physical contact to VSMCs. Conclusions Normal shear stress is a protective factor with its inhibitory effect on EC proliferation. VSMCs induced EC proliferation via TGFβ1 and pAkt pathways by paracrine model.