Avian influenza H5N6 virus not only wreaks economic havoc in the poultry industry but also threatens human health. Strikingly, as of August 2022, 78 human beings were infected with H5N6, and the spike in the number of human infections with H5N6 occurred during 2021. In the life cycle of influenza virus, neuraminidase (NA) has numerous functions, especially viral budding and replication. Here, we found that NA-D272N mutation became predominant in H5N6 viruses since 2015 and significantly increased the viral replication and virulence in mice. D272N mutation in NA protein increased viral release from erythrocytes, thermostability, early transcription, and accumulation of NA protein. Particularly, the dominant 272 residue switch from N to S has occurred in wild bird-origin H5N6 viruses since late 2016 and N272S mutation induced significantly higher levels of inflammatory cytokines in infected human cells. Therefore, comprehensive surveillance of bird populations needs to be enhanced to monitor mammalian adaptive mutations of H5N6 viruses.