In 2023, the first case of dual infection with H10N5 avian influenza virus and seasonal H3N2 influenza virus in humans was reported in Anhui Province, China, marking the first documented cross-species infection of this subtype in humans. This study utilized 1625 H10 genomic sequences to delineate the origin, transmission, and reassortment of the virus. The bayesian evolutionary analysis shows that the virus belongs to a subclade of the H10 North America lineage, which was transmitted into Asia between 2015 and 2017. Reassortment analysis implies that the virus originated from an H10Nx ancestor and obtained the NA gene in Asia from A/duck/Bangladesh/WF-506/2024-like H7N5 donor between 2018 and 2021. Molecular characterization reveals that the HA gene cleavage site of the H10 subtype avian influenza virus infecting humans contains only a single basic amino acid, which is a hallmark of low-pathogenicity viruses. Growth competition assays demonstrate that the H10N5 subtype avian influenza virus exhibits rapid proliferation and sustained high replication capacity in chicken embryos. Additionally, recombinant avian α-interferon (rChIFN-α) exerts relatively weaker inhibitory effects on the proliferation of the H10N5 subtype avian influenza virus in chicken embryos compared with the H9N2 and H6N6 subtypes. The underlying mechanisms of its action require further investigation. Given that the H10 subtype belongs to avian influenza viruses, it has a high affinity for NeuAcα2,3 Gal receptors. These findings underscore the importance of continuous surveillance of the H10N5 subtype avian influenza virus to guard against potential public health risks associated with its potential mutations.