Canine pyometra is a suppurative uterine infection associated with immune dysregulation. This study investigated the role of regulatory T cells (Tregs) and associated factors in the pus accumulation within the canine uterus. Sixteen client-owned intact bitches, eight diagnosed with pyometra and the other eight healthy animals undergoing elective ovariohysterectomy, were enrolled. Blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-coated tubes for flow cytometry. Tissue samples were obtained after ovariohysterectomy and used to examine localization of interleukin (IL)-2 and IL-2Rα as key regulators of Treg functions. Gene expression was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results of flow cytometry analysis revealed a significant increase in the population of Tregs in the uterine tissue and their corresponding decrease in the peripheral blood. This shift is likely reflective of the recruitment of Tregs from the peripheral blood to the decidua in pyometra. There was a marked upregulation in the expression of IL-2 in the uterine tissue. There was dysregulation in the expression of anti-inflammatory cytokines produced by Tregs (such as IL-10) and pro-inflammatory factors secreted by effector T cells (such as RORγt and IL-17A), which gave a deeper insight into the mechanism underlying the immune dysfunction in canine pyometra. Taken together, these observations elucidate the dynamic changes in Tregs and related factors during canine uterine pyometra, thus providing a new perspective on the equilibrium of the uterine immune microenvironment.