Vascular endothelial cell (VEC) forms a monolayer on the smooth muscle cell layer of blood vessel. Thus, substrate stiffness should be considered as one of the most important element in mimicking the physiological condition of VECs. In this research, the effect of substrate stiffness on shear stress induced cellular motility was investigated. VECs are seeded on polydimethylsiloxane (PDMS) substrate with controlled stiffness, ranging from 1.78 ㎫ to 48 ㎪. Shear stress system based on parallel plate flow chamber is utilized to apply laminar shear stress of 15 dyne/㎠. In order to explain the detailed mechanism of cellular migration, expression and distribution of cytoskeleton related pathway consisting RhoA, Rac1 and Cdc42 are examined. In addition, the regulation of syndecan-4 is observed as a mechanoreceptor. Results reveal that there is an optimal stiffness in maximizing the cell motility.