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. The present study 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 after the first dengue vaccination, however the second vaccination provided similar increases (4- to 8-fold) in antibody levels against all types of dengue virus. Thus, the dengue tetravalent DNA vaccine could provide balanced induction of dengue antibody responses even in mice preimmunized with any of the three flaviviruses, at least after the second vaccination.