Influenza vaccines based on conserved antigens could provide cross-protection against infections by multiple subtypes of influenza A virus. Influenza matrix protein 2 (M2) is highly conserved in all influenza A strains. In this study, we deleted the transmembrane domain of the M2 of the avian influenza virus (AIV) A/Chicken/Jiangsu/7/2002 (H9N2) strain to create an M2 without a transmembrane domain, named sM2, which was efficiently expressed in Escherichia coli. The sM2 protein was administered intranasally to mice in combination with chitosan adjuvant three times at an interval of 3 weeks. Three weeks after the last immunization, the mice were challenged with a lethal dose (5 x LD(50)) of A/Chicken/Jiangsu/7/2002 (H9N2) virus, PR8 (H1N1) virus and A/Chicken/Henan/12/2004 (H5N1) virus. The protective immunity of the vaccine was evaluated by determining the survival rates, residual lung virus titers, body weight, and the serum antibody titers of the mice. Nasal administration of 15 microg sM2 in combination with chitosan completely protected mice against the homologous virus and protected 90 and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the sM2 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and that the adjuvant chitosan improved the efficacy of the sM2 vaccine.