H9N2 avian influenza viruses (AIVs) can cross species barriers and expand from birds tomammals and humans. It usually leads to economic loss for breeding farms and poses a serious threat to human health.This study investigated the molecular characteristics of H9N2 AIV isolated from a racing pigeon and its pathogenesis in BALB/c mice and pigeons. Phylogenetic analysis indicated that the H9N2 virus belonged to the Ck/BJ/94-like lineage, and acquired multiple specific amino acid substitutions that might contribute to viral transmission from birds to mammals and humans. A pathogenesis study showed that both mice and pigeons infected with H9N2 virus showed clinical signs and mortality. The H9N2 viruses efficiently replicated in mice and pigeons. In our study, high levels of viral shedding were detected in pigeons, but the infection was not transmitted to co-housed pigeons. Histopathological examination revealed the presence of inflammatory responses in the infected mice and pigeons. Immunohistochemical analysis showed the presence of H9N2 virus in multiple organs of the infected mice and pigeons. Moreover, the infected mice and pigeons demonstrated significant cytokine/chemokine production. Our results showed that the H9N2 virus can infect mice and pigeons, and can not be transmitted between pigeons through direct contact.