Emergence of avian origin and equine origin canine influenza viruses (CIVs) in Asia and the United States brings important concerns. Humans are in closer and more frequent contact with dogs than other common hosts of influenza. Thus, CIV is a potential threat to human health. However, little is known about the determinants of CIV host tropism or the transmissibility of CIVs to humans. An amino acid change (W222L) was implicated in modifying hemagglutinin receptor binding by CIV. This was tested using reverse genetics, glycan microarray and virus histochemistry. Glycan microarray demonstrated that avian-origin CIV (H3N2-222W) bind predominantly to alpha-2, 3 linked glycans. Virus histochemistry indicated that rH3N2-222L had higher binding affinity with epithelial cilia of canine tracheal tissue and weaker binding with avian tracheal tissue. Ferret infection demonstrated that the avian-origin H3N2 CIV could cause infection and limited to rhinitis, suggesting that CIV could infect humans.