Previously, we found that co‐expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3⁺ cells present in CD25^high, CD25^low and even CD25^– subsets of CD4⁺ cells expressed high levels of TNFR2. Consequently, TNFR2‐expressing CD4⁺CD25⁺ Treg included all of the FOXP3⁺ cells present in the CD4⁺CD25^high subset as well as a substantial proportion of the FOXP3⁺ cells present in the CD4⁺CD25^low subset. Flow cytometric analysis of PB identified five‐fold more Treg, determined by FOXP3 expression, in the CD4⁺CD25⁺TNFR2⁺ subset than in the CD4⁺CD25^high subset. In addition, similar levels of FOXP3⁺ cells were identified in both the CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁺CD127^low/− subsets. Furthermore, the CD4⁺CD25⁺TNFR2⁺ subset expressed high levels of CTLA‐4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4⁺CD25⁺TNFR2⁺ cells were anergic and markedly inhibited the proliferation and cytokine production of co‐cultured T‐responder cells. In contrast, CD4⁺CD25⁺TNFR2^– and CD4⁺CD25^–TNFR2⁺ T cells did not show inhibitory activity. As some non‐Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.