Objective The effect of different flow shear stress gradient on the changes of arrangement and shape of endothelial cells was evaluated in order to investigate the effect of shear stress gradient on ECs morphology and function. Method A flow chamber system with gradient shear stress was established, in which the range of shear stress is from 15 dyn/cm2 to 6.6 dyn/cm2(1 dyn=10-5 N), and the shear stress gradient is 1.5 dyn/cm2 and 3 dyn/cm2 respectively. After ECs were subjected to the gradient shear stress for 6 hours, cell angle, cell width length ratio, as well as cell shape index of ECs under the different shear stress gradient were examined. Results The cell angles of ECs were straggling under both 1.5 dyn/cm2 and 3 dyn/cm2 shear stress gradient. The cell width length ratio and cell shape index of ECs were decreased under 1.5 dyn/cm2 shear stress gradient compared with that of 3 dyn/cm2shear stress gradient. Conclusions The ECs show random orientation under the different shear stress gradient. The ECs are trending to stretch and elongate shape under smaller shear stress gradient, and to approach cycloid under larger shear stress gradient.