The ongoing panzootic of H5N1 clade 2.3.4.4b high pathogenicity avian influenza virus (HPAIV) increasingly involves non-traditional hosts such as seabirds and mammals. To assess their role in viral-host dynamics and cross-species transmission, we conducted experimental infections in yellow-legged gulls (Larus michahellis) and American minks (Neogale vison) using HPAIV strains isolated from gulls (H5N1/gull) and minks (H5N1/mink). Infections of gulls with H5N1/gull and minks with H5N1/mink caused viral shedding and high mortality, respectively, and efficient viral transmission from gulls before they developed clinical symptoms. While there was no evidence for H5N1/mink infecting gulls, H5N1/gull subclinically infected minks, followed by neurotropism with a spontaneous emergence of a key mammalian adaptation mutation in the brain, which demonstrates that H5N1 clade 2.3.4.4b spillover events from gulls to minks can lead to fast mammalian adaptation.