The vascular endothelium is exposed to three types of mechanical forces: blood flow-mediated shear stress, vessel-diameter dependent wall tension and hydrostatic pressure. Despite considerable variations of blood pressure in normal and pathological physiology, little is known about the acute molecular and cellular effects of hydrostatic pressure on endothelial cells. Here, we used a combination of quantitative fluorescence microscopy, atomic force microscopy and molecular perturbations to characterize the specific response of endothelial cells to pressure application. We identified a two-phase response of endothelial cells to acute (1 h) vs. chronic (24 h) pressure application (100 mmHg). While both regimes induce cortical stiffening, the acute response is linked to calcium-mediated myosin activation, whereas the chronic cell response is dominated by increased cortical actin density and a loss in endothelial barrier function. GsMTx-4 and amiloride inhibit the acute pressure response, which suggest the sodium channel ENaC as key player in endothelial pressure sensing. The described two-phase pressure response may participate in the differential effects of transient changes in blood pressure and hypertension.