In this study, we found an effective and novel therapeutic approach to atopic dermatitis (AD) therapy via treatment with a canine adipose tissue stem cell (cATSC) extract. We determined that the therapeutic application of cATSC-derived interleukin 10 (IL10) and transforming growth factor β1 (TGFβ1) effectively modulated the overloaded immune response after the induction of AD. In addition, we investigated the molecular role of the cATSC extract during AD treatment. Dogs with naturally occurring AD that was treated at Seoul National University Veterinary Teaching Hospital was enrolled in this study. Owner consent was obtained for privately owned dogs before enrollment. We prepared a primary fat-derived cATSC extract that contained various functional factors, including IL10 and TGFβ1, as a treatment for AD. We found that the cATSC extract significantly ameliorated the pathological symptoms of canine AD. The cATSC extract secreted the immunomodulatory cytokines IL10 and TGFβ1, which modulated the overloaded immune response after the induction of AD. Moreover, these immunomodulatory cytokines modulated AD-induced inflammation and inactivated the pathological signals IL6, INFγ, iNOS, eNOS and Nox4. Additionally, these cytokines protected against apoptotic keratinocyte degeneration. This study demonstrated the novel therapeutic efficacy of the cATSC extract during successive AD treatments, which suggests a potential therapeutic use for human AD patients.