An avian-like H3N2 influenza A virus (IAV) has recently caused sporadic canine influenza outbreaks in China and Korea, but the molecular mechanisms involved in the interspecies transmission of H3N2 IAV from avian to canine species are not well understood. Sequence analysis showed that residue 222 in the hemagglutinin is predominantly tryptophan (W) in closely related avian H3N2 IAV but was leucine (L) in canine H3N2 IAV. In this study, reassortant viruses rH3N2-222L (canine-like) and rH3N2-222W (avian-like) with hemagglutinin mutation L222W were generated using reverse genetics to evaluate the significance of the L222W mutation on receptor binding and host tropism of H3N2 IAV. Compared to rH3N2-222W, rH3N2-222L grew more rapidly in MDCK cells and had significantly higher infectivity in primary canine tracheal epithelial cells. Tissue binding assays demonstrated that rH3N2-222L had a preference for canine tracheal tissues rather avian tracheal tissues, whereas rH3N2-222W favored slightly avian rather canine tracheal tissues. Glycan microarray analysis suggested both rH3N2-222L and rH3N2-222W bound preferentially to alpha-2, 3 linked sialic acids. However, the rH3N2-222W had more than 2-fold less binding affinity than rH3N2-222L to a set of glycans with Neu5Aca2-3Galb1-4(Fuca-)-like or Neu5Aca2-3Galb1-3 (Fuca-)-like structures. These data suggest the W to L mutation at position 222 of the hemagglutinin could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinities of the hemagglutinin to specific receptors with Neu5Aca2-3Galb1-4(Fuca-) or Neu5Aca2-3Galb1-3(Fuca-)-like structures that are present in dogs