Naturally arising CD4⁺CD25⁺ regulatory T (T_R) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4⁺CD25^– T cells also possesses regulatory activity. Programmed death‐1 (PD‐1) is a new member of the CD28/CTLA‐4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4⁺CD25^–PD‐1⁺ T cells in the spleen of naive mice that constitutively expressed CTLA‐4 and FoxP3 and was hypoproliferative in response to anti‐CD3 antibody stimulation in vitro. However, the CD4⁺CD25^–PD‐1⁺ T cells uniquely produced large amounts of IL‐4 and IL‐10 in response to anti‐CD3 and anti‐CD28 mAb stimulation, unlike the CD4⁺CD25⁺ T_R cells. The CD4⁺CD25^–PD‐1⁺ T cells exhibited a suppressor activity against the proliferation of anti‐CD3 antibody‐stimulated CD4⁺CD25^–PD‐1^– T cells in vitro, which was partially abrogated by anti‐CTLA‐4 mAb, but not by anti‐IL‐10 or anti‐PD‐1 mAb. Remarkably, the CD4⁺CD25^–PD‐1⁺ T cells inhibited the development of colitis induced by adoptive transfer of CD4⁺CD45RB^high T cells into C.B17‐scid/scid mice, albeit to a lesser extent than CD4⁺CD25⁺ T_R cells, in a CTLA‐4‐dependent manner. These results indicate that the CD4⁺CD25^–PD‐1⁺ T cells contain substantial amounts of T_R cells that are involved in the maintenance of peripheral tolerance.