We previously demonstrated that a DNA-based tetravalent dengue vaccine can induce balanced neutralizing antibody responses against all four types of dengue virus (DENV1–4), using naïve mice. On the other hand, individuals receiving the dengue vaccine may have previously been infected with dengue virus (DENV1–4) or vaccinated with other fl avivirus vaccines. Thus, we investigated the effect of preimmunity against fl aviviruses on immunogenicity of a dengue tetravalent DNA vaccine in mice. Mice preimmunized with DENV2 developed higher antibody levels against DENV2 than other types of dengue virus after the fi rst dengue vaccination. However, the second vaccination provided similar increases (4- to 8-fold) in antibody levels against all types of dengue virus, compared with non-preimmunized mice. Preimmunization with yellow fever or Japanese encephalitis vaccines did not affect the immunogenicity of the tetravalent vaccine, except for slight and occasional increases in antibody levels against certain types of viruses. Thus, the dengue tetravalent DNA vaccine could provide balanced induction of dengue antibody responses even in mice preimmunized with any of the three fl aviviruses, at least after the second vaccination.