Highly pathogenic avian influenza H5N1 virus has spread to over 1,080 dairy farms across 18 states in the United States, resulting in 41 human infections and posing serious risks to both animal and public health. To address these risks, a hemagglutinin-based mRNA-lipid nanoparticle vaccine was developed, and its safety, immunogenicity, and protective efficacy in high-yielding lactating dairy cows were evaluated. The vaccine was well tolerated, had no adverse effects on health or milk production, and induced strong antibody responses. Two weeks after the second immunization, all the immunized cattle were fully protected against a high-dose H5N1 virus challenge. Notably, two-thirds of the cattle were still completely protected even at the 19th week after the first vaccination, when their serum antibody levels were very low. These data demonstrate that the mRNA vaccine confers robust, lasting protection against H5N1 virus in lactating dairy cows, providing a foundation for clinical trials.