CD4⁺CD25⁺ regulatory T cells (T_reg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of T_reg cells requires the use of reliable surface receptors that are selectively up‐regulated in T_reg cells. On the basis of global gene expression studies, we identified neuropilin‐1 (Nrp1) as a specific surface marker for CD4⁺CD25⁺ T_reg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4⁺CD25⁺ T_reg cells independently of their activation status. In contrast, Nrp1 expression is down‐regulated in naive CD4⁺CD25^– T cells after TCR stimulation. Furthermore, CD4⁺Nrp1^high T cells express high levels of Foxp3 and suppress CD4⁺CD25^– T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish T_reg cells from both naive and recently activated CD4⁺CD25⁺ non‐regulatory T cells.