In vivo electroporation-mediated gene therapy in large animals is gaining ground as one of the most important means for non-viral gene therapy. This review focuses on the novel aspects of reversible electroporation as applied to large animals, improvement of electroporation delivery technique, and development of electroporation-based vaccines. In regard to large animals, we have summarized the initial use of electroporation-mediated antineoplastic gene therapy in humans, vaccination in monkeys, reversing and preventing cachexia in dogs, and increases growth rate and piglet survival in pigs. Novel techniques incorporating electroporation, including ex vivo manipulations, electron avalanche transfection, and electrosonoporation illustrate evolving modifications. Specific alterations of electroporation parameters and DNA formulations along with ideas of enhancing gene transfection efficiency are provided in addition to a discussion of some of the current limitations of electroporation-mediated gene therapy.