Regulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Little is known, however, about the nature of the physiological target antigens for CD4⁺ regulatory T (Treg) cells. Here we report the identification of the LAGE1 protein as a ligand for tumor-specific CD4⁺ Treg cell clones generated from the tumor-infiltrating lymphocytes (TILs) of cancer patients. Phenotypic and functional analyses demonstrated that they were antigen-specific CD4⁺ Treg cells expressing CD25 and GITR molecules and possessing suppressive activity on the proliferative response of naive CD4⁺ T cells to anti-CD3 antibody stimulation. Ligand-specific activation and cell-cell contact were required for TIL102 Treg cells to exert suppressive activity on CD4⁺ effector cells. These findings suggest that the presence of tumor-specific CD4⁺ Treg cells at tumor sites may have a profound effect on the inhibition of T cell responses against cancer.