Dogs are considered mixing vessels for influenza viruses, posing a pandemic potential via viral reassortment. Our previous studies indicated that the avian-origin H3N2 canine influenza virus (A/canine/Zhejiang/1/2010, abbreviated C1) is virulent in canine and mice. Furthermore, we found that the HA and NA genes of C1 share a close genetic relationship with an H3N2 avian influenza virus (A/duck/Shanghai/06/2009, abbreviated D6), but they exhibit distinct pathogenicity. However, the understanding mechanisms remain unclear. In the present study, we explored the genetic determinants that contribute to the different pathogenicity between the C1 and D6. By using the reverse genetics approaches, we rescued several single-gene and position-substituted reassortant viruses based on the C1. The replication in Madin-Darby canine kidney cells and pathogenic trial in mice showed that the neuraminidase (NA) gene played a critical role in C1 virulence. Further analysis demonstrated that the K54E and S154P mutations in NA significantly reduced NA enzymatic activity, impairing viral release from infected cells. Consequently, these mutant viruses lost their ability to infect mice. Overall, our findings identify two novel virulence determinants in NA and elucidate the mechanisms behind the distinct pathogenicity between the C1 and D6 in mice. These results may provide some new targets for H3N2 influenza virus vaccines and antiviral drug development.