The first case of human infection with H5N6 AIVs was reported in southwest China’s Sichuan Province in 2013 (Pan et al., 2016). Results of epidemiological surveillance show that the viruses have recently been isolated from humans (Shen et al., 2016), domestic poultry (Bi et al., 2015; Butler et al., 2016; Du et al., 2017; Li et al., 2017), pigs (Li et al., 2015), environmental samples (Yuan et al., 2016), cats (Yu et al., 2015), and wild birds (Bi et al., 2016b) and resulted in heavy losses in the poultry industry. However, the pathogenicity and transmissibility of H5N6 AIVs have remained unclear. In the current research, we systematically investigated the pathogenicity, transmissibility and the host immune-related gene in the target tissues of infected chickens when challenged by those of wild bird-origin H5N6 AIVs. Our findings provide insights into understanding the host innate immune response of chickens to infection with different pathogenicities of wild bird-origin H5N6 AIVs.