Highly pathogenic avian influenza H5N1 poses an increasing public health risk, particularly following its spillover into dairy cows and associated human infections in the U.S. since March 2024. Here, we systematically identified critical PB2 mutations emerged during avian-to-cattle transmission and subsequent adaptation in cattle, notably PB2 M631L, which conferred pathogenicity in mice comparable to the well-characterized PB2 E627K mutation. Retrospective analysis reveals that PB2 631L also circulated in avian and human H5N1 strains during the 2013-2014 outbreaks in Cambodia and Vietnam. Additional adaptive mutations include established markers (E627K, Q591R, D701N), and novel variants (I647V, G685R, K736R). These mutations enhance polymerase activity by improving the utilization of both bovine and human ANP32A proteins, thereby increasing viral fitness and pathogenicity in mammals. The convergence of these adaptations highlights the elevated zoonotic risk of cattle-adapted H5N1 viruses and underscores the urgent need for heightened surveillance across avian and mammalian hosts.