Regulatory T (Treg) cells represent a subpopulation of suppressor CD4+ T cells critically involved in the establishment of peripheral tolerance through the inhibition of effector T (Teff) cells and the suppression of the immune-mediated tissue destruction toward self-antigens. Treg generation, their suppressive properties and also Treg-Teff cell interactions could be modulated at least in part by programmed cell death-1 (PD-1) expression on their surface and through binding between PD-1 and programmed cell death ligand-1 (PD-L1). Defects involving PD-1 and Tregs can lead to the development of pathological conditions, including autoimmune disorders or promote cancer progression by favoring tumor evasion from the host immune response. At the same time, PD-1 and Tregs could represent attractive targets for treatment, as demonstrated by the therapeutic blockade of PD-L1 applied for the management of different cancer conditions in humans. In the present Review, we focus specifically the role of PD-1/PD-L1 on Treg development and activity.