--- - |2 Summary Regulatory T (Treg) cells are critical suppressors of inflammation and are thought to exert mainly deleterious effects in cancers. In colorectal cancer (CRC), Foxp3+ Treg accumulation in tumors was associated with poor prognosis. Hence, we examined the circulating Treg cells in CRC patients. Compared to controls, CRC patients presented mild upregulations in CD4+CD25+/hi T cells and in the more canonical CD4+CD25+/hiFoxp3+ Treg cells in peripheral blood mononuclear cells. Both of these Treg populations could be roughly divided into lymphocyte activation gene 3 negative T cell immunoglobulin and mucin‐domain containing‐3 negative (LAG3−TIM3−) and LAG3+TIM3+ subsets. In CRC patients, the LAG3+TIM3+ subset represented approximately half of CD4+CD25+/hi T cells and greater than 60% of CD4+CD25+/hiFoxp3+ Treg cells, which was significantly more frequent than in healthy controls. Compared to the LAG3−TIM3− CD4+CD25+/hi T cells, the LAG3+TIM3+ CD4+CD25+/hi T cells presented considerably higher transforming growth factor‐β and slightly higher interleukin (IL)‐10 secretion, together with higher cytotoxic T‐lymphocyte associated protein 4 and Foxp3 expression levels. Notably, macrophages following incubation with LAG3−TIM3− CD4+CD25+/hi T cells and LAG3+TIM3+ CD4+CD25+/hi T cells displayed different characteristics. Macrophages incubated with LAG3+TIM3+ CD4+CD25+/hi T cells presented lower expression of major histocompatibility complex class II, CD80, CD86, and tumor necrosis factor‐α but higher expression of IL‐10, than macrophages incubated with LAG3−TIM3− CD4+CD25+/hi T cells. Together, our investigations demonstrated that CRC patients presented an enrichment of circulating Treg cells, in which the LAG3+TIM3+ subset exhibited more potent expression of inhibitory molecules, and furthermore, the LAG3+TIM3+ Treg cells could suppress the proinflammatory activation of macrophages more potently than the LAG3−TIM3− Treg cells. - Clinical and Experimental Pharmacology and Physiology, Volume 45, Issue 10, Page 1002-1009, October 2018.