Delivery of electric field pulses, i.e. electroporation (EP), to tissues has been shown to have a blood flow modifying effect. Indeed, the diameter of blood vessels exposed to EP is immediately reduced resulting in blood flow abrogation, followed by an increase in vascular permeability. The main cause of the increased permeability remains unknown. The aim of this study was to determine whether the in vivo effects of EP on permeability of blood vessels are linked to the permeabilization of endothelial cells' membrane (EC) and/or disruption of cell-to-cell junctions. We used a dorsal window chamber model in C57Bl/6 mice coupled with multiphoton microscopy and fluorescently labelled antibodies against PECAM-1 (CD31) to visualize endothelial cell-to-cell junctions. Clinically validated EP parameters were used and behavior of cell-to-cell junctions, in combination with leakage of 70 kDa fluorescein isothiocyanate labelled dextran (FD), was followed in time. After EP, a constriction of blood vessels was observed and correlated with the change in the shape of ECs. This was followed by an increase in permeability of blood vessels for 70 kDa FD and a decrease in the volume of labelled cell-to-cell junctions. Both parameters returned to pre-treatment values in 50% of mice. For the remaining 50%, we hypothesize that disruption of cell-to-cell junctions after EP may trigger the platelet activation cascade. Our findings show for the first time in vivo that alterations in cell-to-cell junctions play an important role in the response of blood vessels to EP and explain their efficient permeabilization.