TNFR2 is predominantly expressed by a subset of human and mouse CD4+CD25+FoxP3+ Tregs. In this study, we characterized the phenotype and function of TNFR2+ Tregs in peripheral lymphoid tissues of normal and tumor bearing C57BL/6 mice. We found that TNFR2 was expressed on 30% −40% of peripheral activated/memory subset of Tregs that were most highly suppressive. In contrast, TNFR2− Tregs exhibited the phenotype of naïve cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4+CD25−TNFR2+ cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2+ Tregs was considerably more potent than that of reportedly highly suppressive CD103+ Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2+ Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity which may account for tumor-infiltrating lymphocyte mediated immune evasion by tumor.