We recently identified CD8+ CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+ CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+ CD122+ cells, whereas other cytokines were similarly expressed in CD8+ CD122+ cells and CD8+ CD122− cells. Suppression of both proliferation and IFN-γ production by CD8+ CD122− cells by CD8+ CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-β Ab. When IL-10 was removed from the conditioned medium from CD8+ CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+ CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+ CD122+ cells and mediates the regulatory activity of these cells.