Migratory birds are one of the main reservoirs and long-distance transmission vectors of avian influenza viruses (AIVs). A migratory-bird-origin H3N8 subtype AIV (A/wild bird/Huadong/sy17/2024, hereafter named as WD/HDsy17/24) was isolated and identified in Jiangsu Province, Eastern China, in 2024. However, few studies have been conducted on the cross-species transmission potential of H3N8 AIVs from migratory birds. Phylogenetic analysis indicated that the eight gene segments of WD/HDsy17/24 originated from different subtypes of AIV such as H3N8, H1, H5N1, H5N2, and H7N7 and was a multirecombinant virus. WD/HDsy17/24 virus showed a binding affinity to both SAα-2, 3-galactose (Gal) and SA α-2, 6?Gal receptors, relatively weak thermal stability and pH stability. The SPF chicken pathogenicity indicated that the virus only causes mild respiratory symptoms and leads to lung hemorrhage and congestion. The mice pathogenicity indicated that the body weight of infected mice drops to the lowest value on the third day after infection, the lung-to-body ratio significantly increases, and inflammatory or edema lesions appear in the lungs. Moreover, this virus can infect guinea pigs through contact transmission and aerosol transmission routes from infected SPF chickens. After infection, obvious secretions can be seen in the eyes of guinea pigs. In the contact transmission group, viral shedding was detected in the nasal wash of one guinea pig at 6 days postinfection (dpi). In the aerosol transmission group, viral shedding was detected in the nasal washes of one guinea pig at both 10 and 12 dpi and in another guinea pig at 14 dpi. After 21 days, the seroconversion rate of serum antibodies in both groups is 100%. These findings underscore the need for continued surveillance of H3N8 viruses to identify circulating strains that may potentially threaten human health.