Atopic dermatitis (AD) is a chronic and inflammatory disease. According to a recent study, administration of canine MSCs is a potential therapy for immunological diseases. However, most related studies involve short-term experiments and acute atopic dermatitis animal models. Thus, studies of repeated subcutaneous injection of canine MSCs for ameliorating long-term inflammatory skin disorders have not yet been established. In this study, we evaluated the effects of long-term canine amniotic mesenchymal stem cells (cAM-MSCs) and calcineurin inhibitors (CNIs) treatments in mouse AD model for up to 8 weeks and compared the differences in therapeutic effect through canine peripheral blood mononuclear cells (PBMCs). Using a mouse model, we validated the therapeutic impact of cAM-MSCs in comparison to pimecrolimus (Pime), the most widely used CNIs, as a therapy for canine AD. Based on our results, we verified that the cAM-MSC treatment group exhibited substantially lower scores for tissue pathologic alterations, inflammatory cytokines, and dermatologic symptoms than the PBS control group. Importantly, compared with Pime, cAM-MSCs were more effective at preventing wound dysfunction and regulating mast cell activity. Additionally, we confirmed that immune modulation proteins (TGF-β1, IDO1, and COX-2) were increased in the cAM-MSCs treatment group. Furthermore, we examined the immunoregulatory effect of cAM-MSCs through the proliferation of T lymphocytes from activated canine PBMCs. As a result, cAM-MSCs suppressed the proliferative capacity of effector T cells from canine PBMCs more effectively than Pime. In conclusion, this study suggested that the cAM-MSCS could be an effective canine treatment for long-term canine AD through regeneration and immunomodulation.