Abstract: Objective:To explore whether duck-originated H6N1 subtype avian influenza virus was effectively replicable and infectious in humans and swines.Methods:Two strains of duck-originated H6N1 virus DK/Jiangxi/35634/2016 and DK/Guiyang/2462/2007 isolated in the previous period were selected, the receptor-binding characteristics of the viruses were analyzed by receptor binding specificity method, the changes of of viral gene loci were analyzed by whole genome sequencing, and the respiratory tract tissues of humans and swines were infected in vitro.Results:Both strains of H6N1 virus were bound to avian SA α-2, 3Gal receptors.Gene sequencing results showed that the hemagglutinin(HA)cleavage locus was PQIETR/GL, belonging to the molecular characteristics of low-pathogenic AIV influenza virus, HA, NA, PB2 and other major gene loci were not mutated, but the two strains of virus could replicate in alveolar cells and macrophages of human lung tissues, as well as in tracheal epithelial cells, alveolar cells and macrophages of swine lung tissues, and cause cell and tissue structure damages.Conclusion:Duck-originated H6N1 subtype avian influenza virus has the genetic characteristics of avian influenza virus.Although it still binds to avian SA α-2, 3Gal receptors, it has the ability to infect swines and humans across the interspecies barrier.Therefore, the epidemic and evolution of influenza virus H6N1 in poultry should be closely monitored.