Electroporation-based therapies, such as electrochemotherapy and electrogene therapy, result in the disruption of blood vessel networks in vivo and cause changes in blood flow and vascular permeability. The effects of electroporation on the cytoskeleton of cultured primary endothelial cells and on endothelial monolayer permeability were investigated to elucidate possible mechanisms involved. Human umbilical vein endothelial cells (HUVECs) were electroporated in situ and then immunofluorescence staining for filamentous actin, beta-tubulin, vimentin, and VE-cadherin as well as Western blotting analysis of levels of phosphorylated myosin light chain and cytoskeletal proteins were performed. Endothelial permeability was determined by monitoring the passage of FITC-coupled dextran through endothelial monolayers. Exposure of endothelial cells to electric pulses resulted in a profound disruption of microfilament and microtubule cytoskeletal networks, loss of contractility, and loss of vascular endothelial cadherin from cell-to-cell junctions immediately after electroporation. These effects were voltage dependent and reversible because cytoskeletal structures recovered within 60 min of electroporation with up to 40 V, without any significant loss of cell viability. The cytoskeletal effects of electroporation were paralleled by a rapid increase in endothelial monolayer permeability. These results suggest that the remodeling of the endothelial cytoskeleton and changes in endothelial barrier function could contribute to the vascular disrupting actions of electroporation-based therapies and provide an insight into putative mechanisms responsible for the observed increase in permeability and cessation of blood flow in vivo.