H5N1 influenza outbreaks have been reported on more than 1,070 dairy farms across 17 states in the US. Damage to the mammary gland and high levels of virus in milk were common features of the infected cattle, but it is unclear how the virus initially invades the mammary glands, and no control strategy is currently available. Here, we found that cattle oral tissues support H5N1 virus binding and replication, and virus replicating in the mouth of cattle transmitted to the mammary glands of dairy cattle during sucking. We also found that an H5 inactivated vaccine or a hemagglutinin-based DNA vaccine induced sterilizing immunity in cows against challenges with different H5N1 viruses. Our study provides insights into H5N1 virus transmission and control in cattle.