Mutations in the polymerase basic 2 (PB2) gene of avian influenza viruses are important signatures for their adaptation to mammalian hosts. Various adaptive mutations have been identified around the 627 and nuclear localization sequence (NLS) domains of PB2 protein, and these mutations contribute to the replicative ability of avian influenza viruses. However, few studies have focused on adaptive mutations in other regions of PB2. In this study, we investigated the functional roles of the D253N mutation in PB2 in an H9N2 virus. This mutation was found to affect an amino acid residue in the middle domain of the PB2 protein. The virus with the D253N mutation showed higher polymerase activity and transiently increased viral replication in human cells. However, the mutant did not show significant differences in viral replication in the respiratory tract of mice upon infection. Our results supported that the D253N mutation in the middle domain of PB2, similar to mutations at the 627 and NLS domains, specifically contributed to the replication of avian influenza viruses in human cells.