Cell membranes can be transiently permeabilized under the application of electric pulses. This process, called electropermeabilization or electroporation, allows hydrophilic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. The method is nowadays used in clinics to treat cancers. Vaccination and gene therapy are other fields of application of DNA electrotransfer. A description of the mechanisms can be assayed by using different complementary systems with increasing complexities (models of membranes, cells cultivated in 2D and 3D culture named spheroids, and tissues in living mice) and different microscopy tools to visualize the processes from single molecules to entire animals. Single-cell imaging experiments revealed that the uptake of molecules (nucleic acids, antitumor drugs) takes place in well-defined membrane regions and depends on their chemical and physical properties (size, charge). If small molecules freely cross the electropermeabilized membrane and have a free access to the cytoplasm, larger molecules, such as plasmid DNA, face physical barriers (plasma membrane, cytoplasm crowding, nuclear envelope) which reduce transfection efficiency and engender a complex mechanism of transfer. Gene electrotransfer indeed involves different steps that include the initial interaction with the membrane, its crossing, transport within the cytoplasm, and finally gene expression. In vivo, additional very important effects of electric pulses are present such as blood flow modifications. The full knowledge on the way molecules are transported across the electropermeabilized membranes and within tissues is mandatory to improve the efficacy and the safety of the electropermeabilization process both in cell biology and in clinics.