Increased populations of regulatory T cells (Tregs) impair anti-tumor immunity. Recently, the transcription factor Foxp3 has been reported to play a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. However, Foxp3 is a nuclear protein and is of limited value in the isolation of Tregs, which is a major reason that many functionally relevant aspects of Treg cells are still unknown. Here, we have characterized CD4(+)CD25(+)CD127(low/)- as the surface marker of regulatory T cells in gastric cancer. 88.1-96.1 of CD25(+)CD127(low/-) T cells expressed Foxp3, the frequency of CD4(+)CD25(+)CD127(low/-) regulatory T cells in the peripheral blood of gastric cancer patients was significantly higher than that in healthy controls. Increased CD4(+)CD25(+)CD127(low/-) regulatory T cells were also present in the tumor microenvironment, such as those found in the ascites fluid, tumor tissue or adjacent lymph nodes. Particularly those Treg cells associated with the TNM stage. In addition, we found that CD4(+)CD25(+)CD127(low/-) Tregs suppressed effector T cell proliferation and also correlated to advanced stage of gastric cancer. Thus, CD4(+)CD25(+)CD127(low/-) can be used as a selective biomarker to enrich human Treg cells and also to perform functional in vitro assays in gastric cancer.