Together with aldosterone an increased plasma Na⁺ concentration stiffens endothelial cells. This process is triggered by epithelial plasma membrane sodium channels (ENaC) located in the luminal membrane of endothelial cells. The increase of extracellular sodium also activates the endothelial Na⁺/K⁺-ATPase, which extrudes excess sodium from the cytosol. Both, ENaC and Na⁺/K⁺-ATPase, present in the plasma membrane are hidden in the endothelial glycocalyx (eGC), a biopolymer layer at the luminal side of blood vessels. The eGC is known to be vascular-protective regulating shear stress mediated NO release and limiting oxidative stress. In addition, the negatively charged eGC preferentially buffers sodium ions. Since the eGC is directly exposed to the blood stream, we hypothesized that the glycocalyx could significantly contribute to sodium homeostasis when extracellular sodium varies in the physiological range. From intracellular sodium measurements in human endothelial cells (EA.hy926) two observations were made: (i) eGC removal by heparinase-treatment facilitates sodium to enter/exit the endothelial cells and (ii) the direction of net sodium movement depends on extracellular sodium concentration and ENaC expression. This was found out by using the sodium channel and pump inhibitors, amiloride and ouabain, respectively. Taken together, we conclude that the eGC serves as a physiological barrier for maintaining the homeostasis of body sodium.