Objective Cell adhesion is an essential process for cell migration. The influence of shear stress on the adhesion of VSMCs was elucidated in a co-culture system and to provide some experimental evident in molecular mechanisms of shear stress-induced cell migration in vascular wall. Methods Using a parallel-plate co-culture flow chamber system, human VSMCs co-cultured with human ECs were exposed to static or laminar shear stress, 15dynes/cm2, conditions for 12 hours, with VSMCs cultured alone at static condition as a control. The adhesion of VSMCs was then assessed with cell adhesion assays. The activation of Phosphatidylinositol-3 kinase (PI3K/Akt) pathway in VSMCs was assessed by western blotting. Results It demonstrated that VSMCs co-cultured with ECs under static condition induced VSMC adhesion, whereas the shear stress applied to EC side for 12h significantly inhibited this process. Western blotting showed that there was a consistent correlation between the level of Akt phosphorylation and the efficacy of shear stress-induced EC regulation VSMC adhesion. Conclusions Our findings indicate that shear stress protect against EC-induced VSMC adhesion. Akt is a major downstream factor of PI3K involved in the process.