The expression of the transcription factor forkhead box P3 (FOXP3) was examined in 62 canine mammary gland tumours via immunohistochemical analysis and association with the known expression of the oestrogen receptor-alpha (ERα), progesterone receptor (PR), c-erbB-2 receptor (HER2/neu), and survival. Positive staining for FOXP3 was present in 22.6% of the tumours and was associated with the histological type. Negative staining for FOXP3 was associated with positive ERα and PR expression (P < 0.001). The expression of FOXP3 in canine mammary gland tumours was significantly associated with the disease-free survival time (P = 0.029). The FOXP3 expression was not an independent prognostic factor in the multivariate analysis, though. The negative expression of FOXP3 in the canine mammary gland tumours was found to be related to histopathologic benignity and a longer survival time in canine mammary gland tumours.