A DNA plasmid expressing both the influenza viral matrix protein (M1) and hemagglutinin (HA) (pHA/M1) as a potential vaccine candidate was investigated. Vaccination with pHA/M1 double insertion plasmids not only induced HA-specific protective antibodies, but also elicited HA and M1-specific CD8 T cell responses. Mice immunized with pHA/M1 dual expressing plasmid showed enhanced HA inhibition titer and increased CD69(+) CD8alpha(+) T cell response compared to groups that received either the vector or a mixture of both pHA and pM1 (pHA+pM1). Furthermore, pHA/M1 immunization resulted in improved protection against both homologous and heterologous challenges.