Purpose: Vascular endothelial cells (ECs) are continuously subjected to mechanical forces such as fluid shear stress, stretching and hydrostatic pressure. The effect of hydrostatic pressure on EC responses has not been fully understood compared to that of the other two stimuli. The purpose of this study is to assess mechanical responses of ECs to these mechanical stimuli. Methods: Bovine aortic ECs were exposed to hydrostatic pressure of 50, 100, and 150 mmHg and fluid shear stress of 3 Pa in simultaneous or successive fashion. Immunofluorescence staining of actin filaments and VEcadherin was then performed to observe cell morphology and cell-cell junctions, respectively Results: The results showed that ECs subjected to 50, 100, and 150 mmHg for 24 h elongated without predominant orientation and exhibited multilayered structure, whereas simultaneous application of 50 and 100 mmHg and 3 Pa for 24 h induced marked elongation and orientation of ECs parallel to the direction of flow maintaining monolayer integrity. This monolayer integrity was lost in ECs subjected to 150 mmHg together with 3 Pa. A successive application of 100 mmHg for 24 h followed by 100 mmHg and 3 Pa for 24 h, indicated that the loss of monolayer integrity due to hydrostatic pressure could not be retrieved by the following simultaneous application. Conclusions: It can be concluded that physiological shear stress of 3 Pa is dominant to physiological hydrostatic pressure up to 100 mmHg, importantly suggesting the relative contribution of physiological hydrostatic pressure and fluid shear stress to endothelial monolayer integrity.