Abstract: Objective:To assess the replication efficiency of chicken-originated H6N2 subtype avian influenza virus in MDCK, A549 and isolated human lungs, and to explore the potential of the virus crossing inter-species to infect human. Methods:Two strains of chicken- originated H6N2 avian influenza viruses, A/CK/HZ/260/2017 and A/CK/DG/651/2019, were selected for gene sequencing and analysis of nucleotide homology and genetic evolution of the full-length genome. The solid-phase direct-binding assay was used to analyze the receptor binding characteristics of the two viruses. MDCK, A549 and the human lung tissues were used to assess the replication of the viruses. Results:The two chicken-originated H6N2 virus genes derived from the Eurasian lineages, both HA and NA came from the GD-SZBJ-like clade, and the internal genes mainly derived from the recombinant virus of two different clades of H6N2, including the GD-SZBJ-like clade and the G1291-like clade. The chicken-originated H6N2 viruses were low pathogenic avian influenza viruses that only bound to the avian receptor SAα-2,3Gal, and no mutation in the receptor binding domain of HA was found to favor the binding of the human-like receptor. Two strains of H6N2 subtype avian influenza viruses can replicate in MDCK and A549 cells. Virus isolation and culture in the isolated human lung tissues were positive. The results of immunohistochemical staining showed that A/CK/HZ/260/2017 virus strain in the human lung tissues and part of bronchioles infected with the HZ260 were positive for influenza viral nucleoprotein, and the A/CK/DG/651/2019 virus strain in the human lung tissues was positive for influenza viral nucleoprotein. Conclusion:The chicken-originated H6N2 subtype avian influenza virus is still a low pathogenic avian influenza virus, which only recognizes and binds to avian receptors. However, the virus can replicate effectively in mammal and human lower respiratory tissues without adaptation, indicating that the chicken-originated H6N2 subtype avian influenza virus has the potential to cross inter-species to infect humans.