CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) are critical for the maintenance of peripheral tolerance， and the suppression of autoimmune diseases and even tumors. Although Treg cells are well characterized in humans， little is known regarding their existence or occurrence in ancient vertebrates. In the present study， we report on the molecular and functional characterization of a Treg-like subset with the phenotype CD4-2(+)CD25-like(+)Foxp3-like(+) from a pufferfish (Tetraodon nigroviridis) model. Functional studies showed that depletion of this subset produced an enhanced mixed lymphocyte reaction (MLR) and nonspecific cytotoxic cell (NCC) activity in vitro， as well as inflammation of the intestine in vivo. The data presented here will not only enrich the knowledge of fish immunology but will also be beneficial for a better cross-species understanding of the evolutionary history of the Treg family and Treg-mediated regulatory networks in cellular immunity.