The H12 subtypes of avian influenza viruses (AIVs) are globally prevalent in wild birds, occasionally spilling over into poultry. In this study, we isolated an H12N8 virus from ducks in a live poultry market. Full genomic analysis revealed that the virus bears a single basic amino acid in the cleavage site of the hemagglutinin gene. Phylogenetic analysis revealed that the eight gene segments of the H12N8 virus belong to the Eurasian lineage and the HA gene was clustered with wild bird-originated H12 viruses, with its NP gene showing the highest nucleotide similarity to 2013-like H7N9 viruses. The H12N8 virus replicated effectively in both mammalian and avian cells without prior adaptation. Moreover, the H12N8 virus could infect and replicate in the upper respiratory tract of BALB/c mice without prior adaptation. The H12N8 virus replicated and transmitted inefficiently in both ducks and chickens and hardly triggered high hemagglutination inhibition (HI) antibody titers in the inoculated and contact animals. These results suggest that the wild bird-origin H12N8 virus has reassorted with viruses circulating in domestic poultry, but it inefficiently replicates and transmits in avian hosts. Our findings demonstrate that H12N8 AIV has emerged in domestic poultry, emphasizing the importance of active surveillance of AIVs in both wild and domestic birds.