Background – Atopic dermatitis (AD) is a common chronic inflammatory skin disease of humans and dogs. Regulatory T cells (Tregs) are essential controllers of immune homeostasis and have been shown to play a key role in human AD, even though frequencies of Tregs in atopic human patients vary greatly. Only two studies have reported Treg numbers in the peripheral blood of dogs with canine AD (CAD). Objectives – This study aimed to assess the numbers of circulating Tregs in healthy and atopic dogs, and to determine whether Treg numbers correlate with age, sex, disease severity or pre-treatment. Animals – Client-owned dogs including 14 healthy dogs and 35 dogs with CAD. Methods – Expression of Tregs in peripheral blood mononuclear cells was evaluated by flow cytometry. Tregs were phenotypically identified as T cells triple positive for CD4, CD25 and FoxP3. Results – The percentage of circulating CD4+CD25+FoxP3+ Tregs in atopic dogs was increased significantly compared to healthy dogs (mean 2.1% versus 1%, P = 0.002) and correlated with disease severity (Pruritus Scale: r = 0.48, P = 0.003; CADESI-04: r = 0.34, P = 0.044). No significant differences in age or sex were found in either group and pre-treatment had no influence on results for atopic dogs. Conclusions – Data suggest that, as in humans, CD4+CD25+FoxP3+ Tregs may contribute to the pathogenesis of CAD as indicated by an association between Treg frequency and disease severity. Further investigation is required to improve the understanding of the role of Tregs in atopic dogs.