Plasmid DNA synthesizing membrane-anchored or secretory Japanese encephalitis virus (JEV) envelope (E) protein and premembrane protein was delivered to mice by intramuscular injection or gene gun. Intramuscular plasmid immunization induced anti-E antibody responses similar to those associated with commercial JEV vaccine. The gene gun induced less antibody response. The 2 forms of the E protein induced similar antibody titers when administered by the same delivery mode. Both plasmids generated high titers of JEV-neutralizing antibodies, although the titers were lower than those induced by the vaccine. Intramuscular DNA immunization induced T helper 1 (Th1) immune responses, and the gene gun induced Th2 responses. Compared with secretory E protein, the membrane-anchored protein heavily skewed the immune response toward either Th1 or Th2, depending on the route of immunization. In an intracerebral JEV challenge model, plasmid-immunized mice had approximately 60% protection; this was not affected by the form of the E protein or by immunization route.