Simple SummaryAtopic dermatitis (AD) is a chronic inflammatory skin disease associated with systemic inflammation and immune modulation. Previous studies showed that extracellular vesicles from human adipose-tissue-derived mesenchymal stem cells reduced inflammatory cytokines and attenuated AD-like symptoms. In this study, we aimed to investigate the effects of canine ASC-exosomes on canine AD using a Biostir-induced AD mouse model. The study found that cASC-EVs improved AD-like dermatitis, decreased serum IgE, ear thickness, and inflammatory cytokines such as IL-4 and IFN-γ in a dose-dependent manner. The study also conducted in vivo toxicity studies in ICR mice and found no systemic toxicity. miRNA analysis using next-generation sequencing showed anti-inflammatory miRNAs present in cASC-EVs, which suggests a promising cell-free therapy for treating canine AD.AbstractAtopic dermatitis (AD) is a chronic inflammatory skin disease that is associated with systemic inflammation and immune modulation. Previously, we have shown that extracellular vesicles resulting from human adipose-tissue-derived mesenchymal stem cells (ASC-EVs) attenuated AD-like symptoms by reducing the levels of multiple inflammatory cytokines. Here, we aimed to investigate the improvement of canine AD upon using canine ASC-exosomes in a Biostir-induced AD mouse model. Additionally, we conducted in vivo toxicity studies to determine whether they targeted organs and their potential toxicity. Firstly, we isolated canine ASCs (cASCs) from the adipose tissue of a canine and characterized the cASCs-EVs. Interestingly, we found that cASC-EVs improved AD-like dermatitis and markedly decreased the levels of serum IgE, ear thickness, inflammatory cytokines, and chemokines such as IL-4 and IFN-γ in a dose-dependent manner. Moreover, there was no systemic toxicity in single- or repeat-dose toxicity studies using ICR mice. In addition, we analyzed miRNA arrays from cASC-EVs using next-generation sequencing (NGS) to investigate the role of miRNAs in improving inflammatory responses. Collectively, our results suggest that cASC-EVs effectively attenuate AD by transporting anti-inflammatory miRNAs to atopic lesions alongside no toxicological findings, resulting in a promising cell-free therapeutic option for treating canine AD.