The use of small interfering RNA (siRNA) is a blossoming technique for gene regulation. However, its therapeutic potential is today severely hampered by the lack of an efficient means of safely delivering these nucleic acids to the intracellular medium. We report here that a single 10 ns high-voltage electric pulse can permeabilize lipid vesicles and allow the delivery of siRNA to the cytoplasm. Combining experiments and molecular dynamics simulations has allowed us to provide the detailed molecular mechanisms of such transport and to give practical guidance for the design of protocols aimed at using nanosecond-pulse siRNA electro-delivery in medical and biotechnological applications.