Highly pathogenic avian influenza virus (HPAIV) H5N1 of Asian origin continues to circulate in poultry and wild birds, causing considerable concern for veterinary and public health in Asia, Europe and Africa. Natural transmission of HPAIV H5N1 from poultry to humans, resulting in infections associated with high mortality, and from poultry or wild birds to large felids and domestic cats has been reported. Experimental infection of cats with HPAIV H5N1 derived from a human patient resulted in lethal disease. The role of cats in the adaptation of HPAIV H5N1 to mammals and vaccination regimens for the eventual protection of cats, however, remain to be elucidated. Here, it was shown that cats can be protected against a lethal high-dose challenge infection by an inactivated, adjuvanted heterologous H5N6 avian influenza virus vaccine. The challenge HPAIV H5N1 was derived from a naturally infected cat. In non-vaccinated cats, low-dose exposure resulted in asymptomatic infections with minimal virus excretion. As diseased cats can transmit the infection to na?ve contact animals, the epidemiological role of H5N1-infected cats in endemically infected areas as a link between wild birds, poultry and humans needs close inspection, and vaccination of cats should be considered to reduce possible human exposure.