Regulatory T cells (Tregs) in peripheral blood and tumor infiltrating lymphocytes (TILs) play crucial roles in suppressing anti-tumor immune responses in cancer patients, and correlate with clinical outcomes. We identified an important subpopulation, CD13+CD4+CD25hiTreg cells, among CD4+CD25hiTreg cells in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with NSCLC (n = 72) or from healthy donors (n = 30). Flow cytometric analyses were performed to study the expression of cell-surface or intracellular markers on the CD4+CD25hiTreg cells. The immune suppressive function of CD13+CD4+CD25hiTreg cells was evaluated by co-culturing with CD4+CD25-T cells that were activated by PHA. Our data showed that, compared with CD4+CD25(Low/-)T cells, CD13 expression was enriched on CD4+CD25hiTreg cells. The CD13+CD4+CD25hiTreg cells also expressed higher levels of Foxp3, CTLA-4, membrane-bound transforming growth factor beta1 (mTGFbeta1) and B7-H1, and are more suppressive to CD25 expression and proliferation of CD4+CD25-T cells. Additionally, we showed that the expression of Foxp3, CTLA-4, B7-H1, mTGFbeta1 and the secretion of TGFbeta1 and IL-10 on CD13+CD4+CD25hiTreg cells was significantly suppressed by anti-CD13 mAb (WM15), and the ability of these cells to suppress CD25 expression and proliferation of CD4+CD25-T cells was inhibited by WM15 as well. Interestingly, the percentage of CD13+CD4+CD25hiTreg cells among the CD4+CD25hiTreg population increased significantly and correlated with pathological stage in NSCLC: healthy donor (9.84% +/- 2.23%) <stage I (21.64% +/- 2.78%) <stage II (31.86% +/- 3.01%) <stage III (45.64% +/- 6.12%) <stage IV (58.78% +/- 12.89%). Moreover, the percentage of CD13+CD4+CD25hiTreg cells decreased dramatically after surgical removal of tumors. CD13 is a new surface molecule for identifying a CD4+CD25hiTreg cell subpopulation with higher suppressive ability. The percentage of CD13+CD4+CD25hiTreg cells among the CD4+CD25hiTreg cell population correlated with the pathological stage in NSCLC and tumor burden. CD13 represents a potential target to suppress Treg cells in anti-tumor therapy.