Disruption of the intercellular interactions between endothelial cells leads to endothelial dysfunction. The role of nanoparticles in plasma membrane stability, actin cytoskeleton organization and intercellular junctions is unclear. Human umbilical vein endothelial cells were treated with zinc oxide NPs in vitro. Cell shape, adhesiveness and plasma membrane integrity were analyzed by means of optical fluorescence microscopy, scanning electron microscopy and flow cytometry methods. Additionally, lactate dehydrogenase activity assay and annexin V staining were performed. The scanning electron microscopy analysis showed changes in morphology and surface topography. The F-actin organization was typical for migrating cells. Cell membrane damage (significant increase in lactate dehydrogenase release and annexin V staining) was observed in the concentration of ZnO nanoparticles above 30 microg/ml. The relationship between ZnO nanoparticles and endothelial dysfunction was clearly established and the importance of cytoskeleton reorganization and loosening of the continuous endothelial monolayer after nanoparticles exposure has been documented.