In sum, both H5N6 AIVs were highly pathogenic to chickens, caused multiple systemic infections in tissues, and were efficiently and rapidly transmitted in chickens. Those results indicate that H5N6 viruses could be transmitted to domestic poultry, which represents a serious threat to the poultry industry and both human and animal health. Furthermore, the expression profiles of PRRs, proinflammatory cytokines, chemokines, and MHC molecules in the tested tissues of H5N6-infected chickens were involved in a tissue-dependent manner. Lastly, our experiments demonstrated that ZH283 was associated with greater pathogenicity in chickens, for high virus titers appeared in tested tissues early in the infection process and were accompanied by the excessive expression of cytokines. Such data provide new insights into the relationship between the pathogenicity of H5N6 AIVs and host immune responses to them in chickens.