The H6N1 subtype avian influenza virus (AIV) is a zoonotic infectious disease pathogen, which poses a threat to human health. In order to study the possible substitution of H6N1 AIV for mammals, an avian-origin H6N1 virus was successively passaged in mice. The results showed that PB2 (L193H and E627K), PA (S709F) and HA (V127I) proteins had multiple amino acid substitutions. The virulence of the mouse-adapted virus was stronger than that of the wild virus, and it was highly pathogenic to mice. Therefore, continued surveillance of these substitutions in poultry H6N1 viruses is required.