Lower responsiveness and higher susceptibility to tolerance are the two main characteristics of neonatal immunity that limit the efficacy of conventional vaccines administered during this period. Based on the fact that DNA immunization of adult organisms is able to generate protective immune responses, we investigated the ability of a plasmid-(NPV1) encoding nucleoprotein (NP) of A/PR8/34 strain of influenza virus to generate a cellular immune response following intramuscular delivery in neonates. Newborn mice immunized with NPV1 plasmid developed significant cytotoxic immunity, comparable to the immune response displayed by adult mice injected with the same dose of plasmid. Furthermore, mice infected with influenza virus 1 month after completion of immunization showed a significant decrease of virus lung titer between day 3 and 7 after challenge, consistent with the protectivity conferred by specific cytotoxic immunity. Thus, mice immunized as neonates with NPV1 plasmid developed a protective cellular immune response, like the adult mice. Therefore, the strategy of DNA immunization may be considered for the purpose of human vaccination to prevent horizontally and vertically transmitted life-threatening infections in infants or children.