The H7N9 subtype avian influenza virus (AIV) is currently the subtype with the highest number of human infection cases, with a mortality rate of nearly 40 %,40 %, posing a serious threat to public health. We have previously reported that two avian H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit distinct pathogenicity in mice, in which the polymerase proteins cooperatively increased pathogenicity in mice. Still, the enhancement did not reach the level of JTC11. To further investigate the genetic basis of the virulence difference, we constructed a series of mutant viruses using reverse genetics. We found that the combination of NA-N322S or NA-G389D mutations with PB2-E627K was not sufficient to increase the pathogenicity of H7N9 in mice, although it was able to enhance the neuraminidase activity of the virus. However, the NA-N322S and NA-G389D mutations combined with PB2-E627K significantly enhanced H7N9 pathogenicity. In addition, these combined mutations enhanced neuraminidase enzyme activity, thereby enhancing viral replication, inflammatory cytokine expression, and lung damage, ultimately increasing pathogenicity in mice. In conclusion, this study reveals that the virulence in H7N9 is a polygenic trait and identifies new virulence-associated residues (NAN322S+G389D combined with PB2-E627K). These findings not only enhance our comprehension of the molecular mechanisms underlying AIVs pathogenicity in mammals, but also provide early warning information for preventing cross-species transmission of the H7N9 virus and for potential future pandemics.