Based on our previous studies, we show that M gene is critical for viral replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing bat M gene with those from human and swine influenza A viruses. However, the key amino acids of M1 and/or M2 proteins responsible for virus replication and pathogenicity remain unknown. In this study, the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of chimeric H17 bat influenza virus by replacing the PR8 M gene. Further studies revealed that the M1 was more crucial for viral growth and pathogenicity in contrast to the M2, and amino acid residues of M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of M1 and M2 proteins identified in this study might be important for influenza virus surveillance and used to produce live attenuated vaccines in the future. Importance The M1 and M2 proteins influence the morphology, replication, virulence and transmissibility of influenza viruses. Although a few key residues in M1/M2 proteins have been identified, whether other residues of M1/M2 proteins involved in viral replication and pathogenicity need to be discovered. In the background of chimeric H17 bat influenza virus, the Eurasian avian-like M gene from A/California/04/2009 significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 for viral growth and pathogenicity in vitro and in vivo, and the key amino acid residues of M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine application in the future. These findings provide important information for knowledge on the genetic basis of virulence of influenza viruses.