The licensed vaccine against hepatitis B virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the woodchuck experimentally infected with woodchuck hepatitis virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg-alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.