Adaptation of viral polymerase complex comprising PB1, PB2 and PA is necessary for efficient influenza A virus replication in new host species. We found that mutation PA-K356R has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of PA-K356R mutation. Avian H9N2 virus with PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA, and raised viral polymerase activity that resulted in elevated viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutations of PA-K356R with PB2-E627K, a well-known mammalian adaptive marker, in H9N2 virus showed further cooperative increase in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation which along with other mutations such as PB2-E627K might render avian H9N2 viruses adapted for human infection.