The urinary trypsin inhibitor (ulinastatin) is used in the clinic to prevent inflammatory responses in patients undergoing cardiopulmonary bypass (CPB); however, the anti-inflammatory mechanism is unclear. In the current study, we recruited 40 patients undergoing selective cardiac valve replacement surgery; and these patients were randomly divided into two groups (ulinastatin group [UG] and control group [CG]). We collected peripheral blood preoperatively, at the end of CPB, and postoperative days 1 and 3 and analyzed the kinetic changes in regulatory T (Treg) cell subsets. There was no statistically significant difference in the number of CD4(+) T cells between the two groups. The number of CD4(+)CD25(+) Treg cells, especially the suppressive activated Treg (aTreg) subset, was higher in the UG than the CG 1 and 3 days postoperatively. Thus, ulinastatin alleviated the inflammatory response during CPB by inducing the expansion of aTreg cells.