An unprecedented spillover of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b to dairy cows in March 2024 has affected over 700 US dairy herds in addition to spillback to commercial poultry and transmission to agricultural workers. HPAI H5N1 incursion into cows causes severe mastitis and significant milk production losses. To better understand disease pathogenesis and natural immunity in lactating cows we inoculated the hindquarters of the udder with HPAI H5N1 genotype B3.13 via the teat canal to mimic intra-mammary route of infection. Inoculated cows displayed clinical responses consistent with that observed in affected US dairy herds including generalized decrease in milk yield and localized viral shedding and mastitis in the hindquarters. Following resolution of infection at 31 days post-inoculation, H5N1 virus was inoculated into the unaffected forequarters of the same cows. The secondary inoculation did not result in the clinical manifestations seen upon initial viral challenge. This demonstrated that primary infection of the hindquarters induced natural immunity that conferred complete protection from both mastitis and virus replication and shedding in the forequarters of the udder. This study provides the first experimental report on reinfection demonstrating intra-mammary inoculation can generate H5N1 immunity to completely protect the cow mammary gland.