DNA vaccines can induce protective immunity against subsequent viral challenge. However, for some DNA vaccines to be effective when administered intramuscularly, cardiotoxin pretreatment is necessary. In this study, we used the technique of in vivo electroporation to facilitate DNA delivery and elicit an immune response without the use of cardiotoxin. Intramuscular delivery of DNA (pE) encoding the Japanese encephalitis virus (JEV) envelope protein-induced anti-E antibodies only when the injected muscles were pretreated with cardiotoxin. In vivo electrotransfer of pE eliminated the need for cardiotoxin pretreatment and produced higher antibody titer than that induced by conventional intramuscular injection. Moreover, the induced immunity also conferred protection against lethal viral challenge. Interestingly, like intramuscular immunization, in vivo electroporation immunization with plasmid pE generated anti-envelope antibodies that were predominantly of the immunoglobulin G2a (IgG2a) isotype. These results suggest that in vivo electroporation can be used as an efficient gene delivery system for DNA vaccines to provide efficient protection against viral infection.