Regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and immune homeostasis. IL-10 has an important role in maintaining the normal immune state. Here, we show that IL-10-secreting Tregs can be delineated in normal mice as CD4⁺CD25⁻Foxp3⁻ T cells that express lymphocyte activation gene 3 (LAG-3), an MHC-class-II-binding CD4 homolog. Although ≈2% of the CD4⁺CD25⁻ T cell population consisted of CD4⁺CD25⁻LAG3⁺ T cells in the spleen, CD4⁺CD25⁻LAG3⁺ T cells are enriched to ≈8% in the Peyer's patch. They are hypoproliferative upon in vitro antigenic stimulation and suppress in vivo development of colitis. Gene expression analysis reveals that CD4⁺CD25⁻LAG3⁺ Tregs characteristically express early growth response gene 2 (Egr-2), a key molecule for anergy induction. Retroviral gene transfer of Egr-2 converts naïve CD4⁺ T cells into the IL-10-secreting and LAG-3-expressing phenotype, and Egr-2-transduced CD4⁺ T cells exhibit antigen-specific immunosuppressive capacity in vivo. Unlike Foxp3⁺ natural Tregs, high-affinity interactions with selecting peptide/MHC ligands expressed in the thymus do not induce the development of CD4⁺CD25⁻LAG3⁺ Tregs. In contrast, the number of CD4⁺CD25⁻LAG3⁺ Tregs is influenced by the presence of environmental microbiota. Thus, IL-10-secreting Egr-2⁺LAG3⁺CD4⁺ Tregs can be exploited for the control of peripheral immunity.