The antigenic variation of influenza virus represents a major health problem, thus continuous efforts have been made to develop broad-spectrum vaccines against influenza virus. Matrix protein 1 (M1) protein is highly conserved in all influenza A strains. In this study, M1 protein was efficiently expressed in Escherichia coli (E. coli), then purified and used for immunization of BALB/c mice by intranasal drip using chitosan as adjuvant. The M1 protein was administered intranasally to mice in combination with chitosan adjuvant twice at an interval of 3 weeks. Three weeks after the second immunization, the mice were challenged with a lethal dose (5×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, bodyweight, and the serum antibody titers of the mice. The results showed that nasal administration of 100μg M1 in combination with chitosan could not only completely protect the mice effectively against the challenge of the homologous virus but also protect 70% and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the M1 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and the adjuvant chitosan significantly improved the efficacy of the M1 vaccine.