Highly pathogenic avian influenza A H5N1 has recently expanded its mammalian host range; in 2024, genotype B3.13 emerged in U.S. dairy cattle with pronounced mammary tropism. In the past, Influenza A virus immunology has been characterized primarily in respiratory infection models, whereas this study delineates immune responses after intramammary infection. An intramammary H5N1 challenge in Jersey cows in the early dry-off period enabled integration of dose- and compartment-resolved (alveoli versus teat cistern) cytokine and chemokine profiles with peripheral leukocyte dynamics and H5/N1-specific antibody responses. Infection-induced quarter-restricted, monophasic inflammatory networks peaking at 3–7 days post-infection, coordinated peripheral myeloid expansion and IFN-γ–competent lymphocyte activation, and rising antibody titres across quarters.