Highly pathogenic avian influenza A(H5N1) virus exhibits a strong tropism for the bovine mammary gland, challenging our understanding of influenza A virus host range and tissue specificity. We performed experimental studies with an influenza A(H5N1) B3.13 genotype virus in female lactating dairy cattle to define the infectious dose, routes of exposure, and factors linked to morbidity and mortality. Here, we demonstrate that intramammary inoculation with as few as 10 TCID50 establishes a robust infection and shedding of high-titer virus in milk. Despite this low infectious dose, H5N1 does not readily transmit via contaminated milking equipment and close contact with infected animals. High-dose intramammary exposure results in severe disease and mortality, while respiratory and oral exposures are less likely to establish productive infection and associated morbidity. This study challenges current hypotheses of H5N1 transmission on dairy farms, raising important questions about potential agent, host, or environmental cofactors contributing to viral spread.