Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is an important human pathogen. Mouse brain-derived, inactivated JEV vaccines have contributed greatly to the reduction in numbers of JE patients in several countries, including Taiwan. However, mice immunized with the Nakayama strain inactivated vaccine show lower protection against a lethal strain of Beijing-1 JEV than those immunized with the homologous vaccine. DNA vaccine encoding the envelope (E) protein gene appears to provide protection against JEV in the mouse model, but it is not known whether such vaccines would confer cross-protection for mice against different strains of JEV. In this study, we evaluated the ability of pE, a plasmid DNA vaccine encoding the Beijing-1 envelope protein to elicit cross-protective immunity against infection with the homologous Beijing-1 strain and two lethal Taiwanese isolates of JEV, CH2195 and CEN. Our results showed that mice immunized with pE were protected against lethal challenge with Beijing-1 JEV as well as two Taiwanese isolates. In addition, nai;ve mice were cross-protected by passive transfer of sera from immunized animals, indicating the crucial role of humoral immunity in protection. These results demonstrate that JEV DNA vaccine can provide effective protection against infection not only with homologous virus, but also with heterologous virus.