The newly emerged avian influenza A H5N1 Clade 2.3.4.4b can infect dairy cows and shed live virus in their milk. Sporadic cattle-to-human infections have been reported, highlighting the urgent need to understand its pathogenesis in mammals. Using both non-lactating and lactating BALB/c mice, we examined the viral tissue tropism, histopathological damages, and host immune responses upon intranasal inoculation with a reverse-genetic virus constructed based on A/dairy cattle/Texas/24-008749-003/2024 (Cattle-H5N1) and comparing with an older reference Clade 1 virus, A/Vietnam/1194/2004 virus (VNM1194-H5N1). Cattle-H5N1 was highly lethal in mice (mLD