H9N2 influenza virus has been clustered into multiple lineages because of antigenic drift. The continuous rapid evolution of H9N2 virus increases the difficulties of the prevention and control programs. As a major antigenic protein, hemagglutinin (HA) protein has always been of interest, especially amino acid mutations altering viral antigenicity. It has been well-documented that some amino acid mutations in HA of H9N2 avian influenza virus (H9N2 virus) alter the viral antigenicity, but little is reported about how these antibody escape mutations affect antigenic variation. Herein, we identified 15 HA mutations which were potentially relevant to viral antigenic drift, and found that a key amino acid mutation A198V at position 198 in HA, the only nonconservative site in the receptor binding sites, was directly responsible for viral antigenic variation. Furthermore, the rF/HA _A198V virus showed poor cross-reactivity to immune sera from animals immunized with the viruses F/98 (A198), SD/SS/94 (A198), JS/Y618/12 (T198), and rF/HA _A198V (V198) by microneutralization (MN) assay. The A198V substitution in parent virus caused significantly decreased cross-MN titers by enhancing the receptor binding activity, but did not prevent antibody (Ab) binding physically. Additionally, the strong receptor binding avidity increased the NA activity significantly, while prevented the viral release from cells. Moreover, A198V substitution promoted H9N2 virus escape from pAbs-neutralizing reaction in vitro, and slightly affected cross-protection in vivo. Our results suggested that the A198V mutation with strong receptor binding avidity contributed to viral antigenicity and immune escape, and played a key role in the process of adaptive evolution of H9N2 virus.