H3N2 canine influenza viruses are prevalent in Asian and North American countries. During circulation of the viruses in dogs, these viruses are occasionally transmitted to cats. If this canine virus causes an epidemic in cats too, sporadic infections may occur in humans because of the close contact between these companion animals and humans, possibly triggering an emergence of mutant viruses with a pandemic potential. In this study, we aimed to gain an insight into the mutations responsible for inter-species transmission of H3N2 virus from dogs to cats. We found that feline CRFK cell-adapted viruses acquired several mutations in multiple genome segments. Among them, HA1-K299R, HA2-T107I, NA-L35R, and M2-W41C mutations individually increased virus growth in CRFK cells. With a combination of these mutations, virus growth further increased not only in CRFK cells but also in other feline fcwf-4 cells. Both HA1-K299R and HA2-T107I mutations increased thermal resistance of the viruses. In addition, HA2-T107I increased the pH requirement for membrane fusion. These findings suggest that the mutations, especially the two HA mutations, identified in this study, might be responsible for adaptation of H3N2 canine influenza viruses in cats.