The great benefit of aldosterone receptor inhibitors for the cardiovascular system has been shown in clinical studies. However, the target cells and the underlying mechanisms are yet unknown. The epithelial sodium channel (ENaC) in endothelium is regulated by aldosterone and known to be an important mediator of endothelial dysfunction. Its expression and membrane insertion modifies the mechanical properties e.g. the stiffness of endothelial cells. The hypothesis is tested whether aldosterone-induced ENaC expression of the vascular endothelium can be prevented by spironolactone in the long term. Human endothelial cells (HUVEC) were cultured over four weeks/passages in presence of aldosterone (0.1–10nM) with or without spironolactone (0.1–1mM). Detection of ENaC expression by semi-quantitative RT-PCR and immunofluorescence microscopy were performed weekly. In parallel, the mechanical stiffness of the cells was monitored by atomic force microscopy (AFM). We found that endothelial cells four weeks in culture increase ENaC mRNA expression by 17% and ENaC membrane insertion by 43%. In parallel, the mechanical stiffness rises significantly by 2.5 fold. In the presence of spironolactone, however, all these effects were significantly diminished. We postulate that the accumulation of endothelial ENaC over time causes endothelial cells to gradually stiffen, which finally leads to endothelial dysfunction. The fact that spironolactone prevents the gradual rise of ENaC expression could explain the protective effect of mineralocorticoid receptor antagonists observed in patients with cardiovascular pathologies.