Endothelial cells have important functions in balancing body fluids. They represent a distinctly regulated barrier for the diffusion of water and solutes between blood vessel and interstitium. The “energy currency” ATP accumulates intracellularly and is released upon cell damage. It then plays a role in paracrine signalling via purinergic receptors for example in the regulation of vascular tone. ATP is known to improve barrier properties of endothelial cells. Here we investigate the mechanism of this effect in human umbilical vein endothelial cells in vitro. The hydraulic permeability was assessed by diffusion of fluorescently labelled dextran across the cell layer. ATP (10 mM) reduced the hydraulic permeability down to 53% of initial value. We applied electrical cell impedance spectroscopy (ECIS) in a parallel experiment to monitor changes in total transendothelial resistance (TER). The total resistance raised to 180% of initial value. Further analysis of the data revealed that this effect is mainly due to increase of the paracellular resistance with only minor contributions of the cell-to-substrate distance. We conclude that improvement of endothelial barrier function through ATP is mediated by tightening of the paracellular shunt.