Chronic wounds increase the risk of infection and may lead to complications or disease. Although treatment techniques involving topical negative pressure have been used widely to promote wound healing, the relationship between promotion of wound healing and negative pressure remains unclear. In the present study, we studied the effects of hydrostatic pressure (HP) on endothelial cells (ECs) during pressure treatment. We examined the morphologic and functional responses of ECs to HP using an experimental system developed to apply both negative and positive pressure to ECs. Morphologic parameters such as aspect ratio, orientation angle, and tortuosity did not change after exposure to HP for up to 24 h. In contrast, application of HP led to significant changes in cell area and cell density, and the formation of intercellular gaps was observed as early as 3 h before the cell density reached its peak value. We also found HP progressed EC cycle, which remained at rest according to contact inhibition. Although there were some differences with respect to trends in changes in those parameters, positive and negative pressures had similar effects on ECs. Considering the results of this study, we conclude that exposure to HP enhances the proliferation of ECs.