Abstract: To investigate the effect of HA S145N mutation on pathogenicity and antigenicity of H9N2 avian influenza viruses (AIV), the EID50, ELD50, MDT, ICPI, IVPI and the duration of shedding virus from infected 8weekold SPF chickens of different H9N2 AIV strains with HA S145N mutation isolated from Mainland China were determined. The HI and VN activity of monoclonal antibody 2A4 and F6 on different H9N2 AIV isolates were assayed. The determined pathogenicity suggested that the H9N2 AIVs with HA S145N mutation have higher pathogenicity and can cause the death of SPF chickens. The 8weekold SPF chickens infected by H9N2 AIVs with HA S145N mutation shed virus earlier and last for a longer time. Monoclonal antibody 2A4 and F6 can inhibit the hemagglutinin activity of H9N2 AIVs with HA S145N mutation, however it can not neutralize the virus infection on CEF cells. The results showed that the H9N2 subtype AIVs have the tendency to evolve, resulting in the occurrence of mutants which have higher virulence and variable antigenicity. The site (S145) is one of HA protein epitope of the H9N2 subtype AIV. The higher virulent mutants may cause death of chickens and lead to more economic loss. The antigenic mutants may escape from immune clearance, which pose a new challenge to the immunoprophylaxis for the H9N2 avian influenza.