The interaction between migratory birds and domestic waterfowl facilitates viral co-infections, leading to viral reassortment and the emergence of novel viruses. In 2022, samples were collected from duck farms around Poyang Lake in Jiangxi Province, China, which is located within the East Asia-Australasia flyway. Three strains of H4N6 avian influenza virus (AIV) were isolated. Genetic and phylogenetic analyses showed that the isolated H4N6 avian influenza viruses (AIVs) belonged to new genotypes, G23 and G24. All isolated strains demonstrated dual receptor binding properties. Additionally, the isolated strains were able to replicate efficiently not only in avian cells but also in mammalian cells. Furthermore, the H4N6 AIV isolates could infect chickens, with viral replication detected in the lungs and extrapulmonary organs, and could transmit within chicken flocks through contact, with viral shedding detected only in oropharyngeal swabs from chickens in the contact group. Notably, the H4N6 AIV could infect mice without prior adaptation and replicate in the lungs with high viral titers, suggesting that it is a potential threat to humans. In conclusion, this study provides valuable insight into the characteristics of H4N6 strains currently circulating in China.