Airborne transmission plays an important role in dissemination of H9N2 subtype avian influenza virus. Annexin II (A2)-mediated activation of plasminogen (PLG) promotes cleavage of the influenza virus HA protein and viral replication, resulting in enhanced pathogenesis. In this study, airborne transmission competent and defective strains of H9N2 influenza virus, SH7 and SH14, respectively, were used to investigate the effect of A2 on airborne spread. The results showed that A2 protein was increased in SH7 virions compared with SH14 particles, the binding ability of the SH7-infected MDCK cells to PLG was significantly higher than the SH14-infected cells, and influence efficiency of the PLG on replicated ability of SH7 virus was significantly stronger than that of SH14 virus, who spread without airborne route, indicating that the annexin 2 (A2) can bind PLG and contributes to SH7 with high replication ability. Furthermore, the copies of SH7 in the airborne infected chickens under inhibited by 6-AHA were significantly decreased, suggesting that the release of H9N2 avian influenza virus were reduced by inhibiting the conversion of PLG to PL, ultimately resulting in reduced airborne transmission of H9N2 avian influenza virus. In summary, A2-mediated conversion of PLG to PL plays a role in the airborne transmission capacity of H9N2 avian influenza viruses, and this interaction may represent potential targets for prevention and treatment of influenza virus infection.