H6N6 viruses are commonly isolated from domestic ducks and avian-to-swine transmissions of H6N6 viruses have been detected in China. Whether subsequent adaptation of H6N6 viruses in mammals would increase their pathogenicity towards humans is not known. To address this, we generated a mouse-adapted swine influenza H6N6 virus (GDK6-MA) which exhibited greater virulence than the wild-type virus (GDK6). Amino acid substitutions in PB2 (E627K), PA (I38M) and HA (L111F, H156N and S263R) occurred in GDK6-MA. HA H156N resulted in enlarged plaque sizes on MDCK cells and enhanced early stage viral replication in mammalian cells. PA I38M raised polymerase activity in vitro but did not change virus replication in either mammalian cells or mice. These single substitutions had only limited effects on virulence, however, a combination of HA H156N, S263R and PA I38M in the GDK6 backbone led to a significantly more virulent variant. This suggests these substitutions can compensate for the lack of PB2-627K and modulate virulence, revealing a new determinant of pathogenicity for H6N6 viruses in mice, which might also pose a threat to human health.