Depletion of CD4(+)CD25(+)FoxP3(+) Treg using PC61 mAb (anti-murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated approximately 70% of CD4(+)FoxP3(+) cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcgamma receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcgamma receptor (FcgammaR)III(-/-) mice were resistant to Treg depletion. FcgammaRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab-dependent-cellular-cytotoxicity and Ab-dependent-cellular-phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab-dependent-cellular-phagocytosis of CD25(+) cells by WT or FcgammaRIIB(-/-), but not FcgammaRIII(-/-), macrophages. Altogether these data demonstrate the critical role of FcgammaRIII(+) phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.