Helios is a member of the Ikaros transcription factor family and has been reported to be a marker of thymus-derived regulatory T cells (Treg). Helios is an intracellular protein, however, and hence cannot be used as a marker to separate living Tregs. To solve this problem, we generated Helios reporter mice in which Helios+ cells selectively express Venus, a variant of green fluorescent protein. Most of the Tregs in the thymus expressed Helios, whereas its expression was varied in peripheral lymphoid organs. The Helios+ Treg-population was superior in ability to suppress both antigen-specific and TCR-stimulated T cell responses. We also showed that Helios+ Tregs inhibited the cytokine production by T cells more efficiently than Helios- Tregs. We conclude that Helios reporter mouse strain is a useful tool to study function of Helios and that Helios+ Tregs represent the highly suppressive population.