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 (mLD50 =1.48PFU) with broad tissue tropism and produced higher titer in respiratory tissue and multiple extrapulmonary organs than VNM1194-H5N1. In the lungs, Cattle-H5N1 infection of airway epithelium, type II pneumocytes and CD45+ immune cells were at a higher frequency than those of VNM1194-H5N1-infected mice, resulting in severe epithelial destruction and diffuse alveolar damage accompanied by elevated lung and serum pro-inflammatory cytokine/chemokines. Although both H5N1 viruses showed lactating mammary gland tropism, the gland tissue was more severely damaged after Cattle-H5N1 infection with abundant viral antigens expression in glandular cells, associated fat and lymphoid tissues. Furthermore, more suckling mice co-housed with Cattle-H5N1 infected lactating mice were virus-positive (7/30 pups) than VNM1194-H5N1. Brains were heavily infected by Cattle-H5N1, and neurological signs such as body-rolling/spinning, trembling and/or limb paralysis were seen only in Cattle-H5N1 infected mice. The spleen was more severely damaged by Cattle-H5N1 infection, which showed massive viral antigen expression accompanied by severe apoptosis and splenic atrophy, concluding that Cattle-H5N1 is more virulent in mice than VNM1194-H5N1.