Highly pathogenic avian influenza (HPAI) H5N1 hemagglutinin clade 2.3.4.4b was detected in the United States in 2021. These HPAI viruses caused mortality events in poultry, wild birds, and wild mammals. On March 25, 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multi-state investigation into milk production losses.1 Over 200 positive herds were identified in 14 U.S. states. The case description included reduced feed intake and rumen motility in lactating cows, decreased milk production, and thick yellow milk.2,3 The diagnostic investigation revealed viral RNA in milk and mammary tissue with alveolar epithelial degeneration and necrosis and positive immunoreactivity of glandular epithelium. A single transmission event, likely from birds, was followed by limited local transmission and onward horizontal transmission of H5N1 clade 2.3.4.4b genotype B3.13.4 We sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows. Heifers were inoculated by aerosol respiratory route and cows by intramammary route. Clinical disease was mild in heifers, but infection was confirmed by virus detection, lesions, and seroconversion. Clinical disease in lactating cows included decreased rumen motility, changes to milk appearance, and production losses. Infection was confirmed by high levels of viral RNA detected in milk, virus isolation, lesions in mammary tissue, and seroconversion. This study provides the foundation to investigate additional routes of infection, pathogenesis, transmission, and intervention strategies.