Regulatory T cells (Treg) comprise multiple subsets and are important in controlling immunity and inflammation. However, the induction and mode of action of the various distinct Treg subsets remain ill defined, particularly in humans. Here, we describe a human CD8⁺lymphocyte activation gene-3 (LAG-3)⁺CD25⁺FoxP3⁺ Treg subset, which suppresses T cells partly through the secretion of CC chemokine ligand 4 (CCL4), which can inhibit T cell activation by interfering with T cell receptor signaling. CD8⁺ Tregs are expanded by antigen in in vivo-primed donors, and can be detected in pathogen-infected human tissue. This CD8⁺LAG-3⁺CD25⁺FoxP3⁺CCL4⁺ Treg subset thus may play a role in immunoregulation in humans, including infectious diseases.