The prevalence and spread of canine influenza virus (CIV) pose a threat to the health of dogs and humans. Some studies have shown that autophagy is closely related to virus replication, but the exact relationship between CIV replication and autophagy is still unclear. Therefore, this study investigated the effects of autophagy on CIV replication in vitro and in vivo. The data showed that CIV infection significantly caused respiratory tract damage in mice, upregulated the mRNA/protein levels of CIV replication-related genes and autophagy-related genes. In addition, the activation of autophagy by rapamycin (Rapa) significantly intensified the CIV replication and the respiratory tract damage of mice, while the inhibition of autophagy by 3-Methyladenine (3-MA) significantly alleviated these effects. Data of MDCK cells also demonstrated that CIV promoted self-replication through activating autophagy, and the upregulation of AKT/mTOR by insulin significantly inhibited the CIV replication. In summary, this study showed that CIV could promote self-replication by activating AKT/mTOR mediated autophagy, which provides new ideas for the prevention and treatment of canine influenza.