Immune responses can be enhanced or dampened by differential manipulation of Foxp3-expressing CD25⁺CD4⁺ natural regulatory T (Treg) cells versus other naive or activated T cells. By searching for a molecule capable of distinguishing these populations, we here found that natural Treg cells constitutively expressed high amounts of folate receptor 4 (FR4). The expression of FR4 and CD25 also separated antigen-stimulated CD4⁺ non-Treg cells into the FR4^hiCD25⁻ and FR4^loCD25⁺ populations, which were different in proliferation and cytokine secretion upon restimulation. These distinctions showed that antigenic stimulation activated and expanded antigen-specific natural Treg cells as well as effector and memory T cells. Accordingly, FR4^hiCD25⁺CD4⁺ T cells enriched from alloantigen-stimulated T cells suppressed graft rejection. Administration of FR4 monoclonal antibody specifically reduced Treg cells, provoking effective tumor immunity in tumor-bearing animals, whereas similar treatment of normal young mice elicited autoimmune disease. Thus, specific manipulation of FR4^hiCD25⁺CD4⁺ Treg cells helps control ongoing immune responses.