Endothelial cells form the innermost layer of blood vessels. Due to this strategic position endothelial cells control many aspects of vascular function. One of the major characteristics of endothelial function is the ability of the endothelial cells to release vasoactive substances such as nitric oxide (NO), which cause a vasodilatation1. Recently, it was shown that stiffening of the endothelial cell cortex, an actin-rich layer 50-150 nm underneath the cell membrane, leads to a reduced NO release and therefore causes an increase in (systemic) blood pressure2. We identified the endothelial Na+ channel (EnNaC) as major regulator of the cortical stiffness in that its membrane abundance stiffens the cortex: An increased abundance of EnNaC in the cell membrane causes a rise in functional stiffness of the endothelial cell cortex which is accompanied by a decreased NO release3. Strikingly, EnNaC-mediated endothelial stiffening can be reversed by pharmacological or genetic downregulation of the channel. Since the actin-polymerizing agent Jasplakinolide reverses this softening process, a direct interaction between EnNaC and the subcortical actin cytoskeleton is postulated. To identify the underlying mechanisms of this observation, specific components of the cortical actin cytoskeleton were degraded by adding inhibitors for the Rac1 (NSC23766) and RhoA pathway (CT04) as well as the Arp 2/3 complex (CK548). Under these conditions, the mechanical stiffness of endothelial cells (EA.hy 926) was probed with the Atomic Force Microscope. In parallel, EnNaC membrane abundance was studied using Quantum Dot-mediated immunofluorescence microscopy. We were able to show that the inhibitor-induced degradation of the actin cytoskeleton leads to a significant reduction in cortical stiffness of 15.0% for NSC23766, 13.0% using CT04 and 18,3% for CK548. This decrease is accompanied by a significant reduction of EnNaC membrane abundance: 35,6% for NSC23766, 25% for CT04 and 38,2% using CK548. We conclude that the structural dynamics of the outer shell of endothelial cells is caused by functional interaction between the subcortical actin cytoskeleton and EnNaC. This interaction results in a change of the cortical stiffness and is thereby one major component responsible for proper endothelial function. With these results we found a novel mechanism of regulation of nanomechanical properties of endothelial cells. Since this is pivotal for vascular function, our results might lead to novel therapeutical strategies regarding the treatment of endothelial dysfunction and cardiovascular pathologies.