The H3N2 canine influenza A virus (IAV), which originated from an H3N2 avian IAV, has caused outbreaks in North America and Asia. Although H3N2 canine IAV has not been reported to transmit to humans, understanding the molecular basis of receptor tropism changes is important for viral surveillance and zoonotic risk assessment. For avian-like IAVs to spill over into humans, the hemagglutinin (HA) glycoprotein on the viral surface must acquire mutations that enable binding to α2,6-linked sialic acids (SA), the primary receptors for human influenza viruses. Although H3N2 canine IAV preferentially binds to α2,3-linked SA, the primary receptors for avian viruses, repeated mutations during viral replication in dogs may lead to HA substitutions that confer binding affinity to α2,6-linked SA. In this study, we serially passaged conditionally replicating H3N2 canine IAV in α2,3-linked SA-knockout MDCK cells, which predominantly express α2,6-linked SA (human-type cells), to identify mutations in the receptor-binding domain. We then generated recombinant viruses carrying individual mutations using reverse genetics and evaluated their growth properties in human-type cells. The HA1-T138A and HA1-Q226L substitutions significantly enhanced viral replication in these cells. Furthermore, a solid-phase glycan-binding assay demonstrated that these mutations increased binding to α2,6-linked SA to levels comparable to those observed for α2,3-linked SA. Together, these findings indicate that H3N2 canine IAV has the potential to acquire mutations that enable efficient binding to α2,6-linked SA. This study suggests that the HA1-T138A and HA1-Q226L substitutions may play critical roles in the adaptation of H3N2 canine IAV to cells expressing human-type receptors.