Mechanical cell stiffness is a crucial parameter of endothelial function as stiff endothelial cells produce less nitric oxide (NO). A reduced availability of the vasodilator NO results in increased peripheral resistance and the development of high blood pressure. The mineralocorticoid hormone aldosterone is one of the factors that increase mechanical stiffness of endothelial cells. Concomitantly, the hormone is known to upregulate ENaC abundance in the endothelial plasma membrane. Functional blockade of ENaC with amiloride softens the cells indicating that ENaC could be involved in mechanical cell stiffening. Therefore, experiments were designed to test for a direct link between ENaC and mechanical stiffness. To this end we have stably down-regulated the ENaC a-subunit in human vascular endothelial cells (EAhy926) using the RNA interference technique. Mechanical stiffnesses of the ENaC knock-down and wild-type cells were measured by atomic force microscopy (AFM). In principle, a spherical AFM tip (sphere diameter: 10 mm) is pressed onto the apical cell surface and the force quantified that is needed to indent the cell for 200 nm. This force can be directly translated into cell stiffness. We found that stiffness of ENaC knock-down cells was 1.11 ± 0.026 pN/nm (n = 121). This value is 15% less in comparison to that of control cells (1.31 ± 0.029 pN/nm, n = 111; p<0.05). ENaC knock-down cells used for the experiments were mixed clones with a mean 50% reduction in ENaC mRNA shown by RT-PCR. Taking this into account, a complete knock-down of the ENaC should lead to a 30% decrease in mechanical stiffness. Such a decrease in cell stiffness is known to lead to a 184% increase in NO production as shown previously (Oberleithner, PNAS 2009). Thus, ENaC, endogenously expressed in endothelium and regulated by aldosterone, influences the mechanical properties of endothelial cells and therefore, may have a significant impact on peripheral resistance and blood pressure.