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Among several mechanisms that allow tumours to disarm the host immune system and thus to evade or suppress protective anti-tumour immunity, an important role for CD4+CD25+FoxP3+ regulatory T cells (Tregs) has emerged. Numerous studies in humans have demonstrated increased Tregs in patients with carcinomas of the breast, lung, and pancreas, and this increased Treg has been correlated with poor prognosis. This study was performed (1) to investigate the percentage of Tregs in total lymphocytes of the peripheral blood in 12 canine patients with B cell lymphoma and (2) to investigate the change in the percentage of Tregs in canine lymphoma of different clinical tumour stages. On the flow cytometric analysis, the relative and absolute numbers of Tregs were significantly increased in 12 canine patients with B-cell lymphoma compared to five healthy beagles included in this study, and the greatest increases in the relative and absolute number of Tregs occurred in two dogs with more advanced World Health Organization clinical stages with bone marrow involvement compared to those in less advanced tumour stages without bone marrow involvement. This study provides basic information regarding the negative role of Treg recruitment in canine lymphoma patients and highlights the potential value of Treg levels as prognostic indicators in canine cancer patients.