Novel H5N8 highly pathogenic avian influenza viruses (HPAIVs) bearing the clade 2.3.4.4b HA gene have been widely spread through wild migratory birds since 2020. One H5N8 HPAIV (A/Wild bird/Cixi/Cixi02/2020; hereafter Cixi02) was isolated from migratory birds in Zhejiang Province, Eastern China in 25 November 2020. However, its pathogenicity in avian and mammal remains unknown. Hemagglutinin gene genetic analysis indicated that Cixi02 virus belonged to the branch II of H5 clade 2.3.4.4b originated from Iraq in May 2020. Cixi02 virus showed a binding affinity to both SA α-2, 3-galactose (Gal) and SA α-2, 6 Gal receptors, good pH stability, thermostability, and replication ability in both avian and mammal cells. The poultry pathogenicity indicated that Cixi02 virus was lethal to chickens. Moreover, the mammalian pathogenicity showed that the 50% mouse lethal dose (MLD50 ) is 2.14 lgEID50 /50 μL, indicating a high pathogenicity in mice. Meanwhile, Cixi02 virus was widely detected in multiple organs, including heart, liver, spleen, lung, kidney, turbinate, and brain after nasal infection. In addition, we found high level gene expressions of TNF-α, IL-12p70, CXCL10, and IFN-α in lungs, IL-8 and IL-1β in brains, and observed severe histopathological change in lungs and brains. Collectedly, this study provided new insights on the pathogenic and zoonotic features of an H5N8 subtype AIV isolated from migratory birds.