Canine atopic dermatitis is a common allergic skin disease in dogs caused by defects in immunological and epidermal barriers. Claudin-1, a protein present in tight junctions, is associated with many skin conditions. Our aim was to investigate expression levels of claudin-1 and atopic signature cytokines in atopic and normal dog skins. The affected skin showed significantly decreased intensity and epidermal distribution of claudin-1 compared to the normal control skin (P < 0.05). The CD3+/CD4+ and CD3+/CD8+ T cell subsets, eosinophils, and neutrophils infiltrated the affected epidermis and dermis. The CD3+/CD4+ T cell subsets in lesional skin were significantly higher than CD3+/CD8+ T cell subsets. CLDN-1 mRNA expression was markedly downregulated in the lesional skin, while IL-17A was upregulated in both lesional and nonlesional skin. IL-4 and IL-31 mRNA was upregulated in the lesional skin, and TNF-α mRNA was significantly upregulated in the nonlesional skin (P < 0.01 and P < 0.05, respectively). Correlation-based hierarchical clustering showed that CLDN-1 expression was closely clustered with IL-31 but loosely clustered with L-4, IL-10, IL-13, and IL-17A. We suggest that the alteration of claudin-1 in atopic dogs may disrupt the skin barrier and allow an influx of allergens, inciting inflammatory cytokine responses.