The adoptive transfer of regulatory Foxp3⁺ T (Treg) cells has been shown in various animal models to prevent inflammatory immune and autoimmune diseases. Translation into therapeutic applications, however, is hindered by the lack of suitable techniques and markers. CD25, commonly used to isolate Treg cells from mice, has only limited value in humans as it is also present on proinflammatory CD4⁺ effector cells. Here we show that clean populations of human Foxp3⁺ Treg cells can be obtained with antibodies directed against CD49d. The marker is present on proinflammatory peripheral blood mononuclear cells but is absent on immune-suppressive Treg cells. Depletion with α-CD49d removes contaminating interferon-γ (IFN-γ)– and interleukin-17 (IL-17)–secreting cells from Treg preparations of CD4⁺CD25^high cells. More importantly, in combination with α-CD127 it allows the isolation of “untouched” Foxp3⁺ Treg (ie, cells that have not been targeted by an antibody during purification). The removal of CD49d⁺/CD127⁺ cells leaves a population of Foxp3⁺ Treg virtually free of contaminating CD25⁺ effector cells. The cells can be expanded in vitro and are effective suppressors both in vitro and in vivo. Thus, CD49d provides access to highly pure populations of untouched Foxp3⁺ Treg cells conferring maximal safety for future clinical applications.