DNA vaccination is an effective means of eliciting both humoral and cellular immune responses. The hemagglutinin (HA) surface protein of influenza A virus is a major target of protective antibody responses induced by virus infection or by vaccination and is widely considered to be the antigen of choice for an influenza vaccine. Cytotoxic T lymphocyte (CTL) responses directed against the conserved nucleoprotein (NP) are thought to play an important role in clearing virus and promoting survival and recovery from influenza. In this study, we developed a novel DNA vaccine approach using a chimeric plasmid consisting of the HA of H5N1 influenza virus in which an MHC class I-restricted NP-specific CTL epitope (NP147-155) was inserted. Immunogenicity and antiviral efficacy of this vaccine was assessed in mouse models. A similar level of HA expression was achieved in 293T cells transfected with pHA/NP(147-155) compared to that with pHA. Besides eliciting the specific anti-HA antibody responses, vaccination using pHA/NP(147-155) in mice induced NP epitope-specific CD8(+) T cell responses, which are generally not inducible by vaccination with pHA alone. After H5N1 influenza virus challenge, BALB/c mice vaccinated with pHA/NP(147-155) exhibited reduced inflammation severity and lung viral titers compared to those vaccinated with pHA. Our work may contribute to improvement of HA-based influenza DNA vaccines.