CD4+CD25+FOXP3+ regulatory T cells (Tregs) constitute a heterogeneous lymphocyte subpopulation essential for curtailing effector T cells and establishing peripheral tolerance. Hence, Tregs have become a key target for immunotherapies in autoimmunity and transplantation. Calcineurin inhibitors (CNIs) are among the most effective agents in controlling effector T cell responses in humans, but they also reduce the size of the Treg pool. The mechanisms responsible for this negative effect and the functional consequences remain to be elucidated. Here we characterize the Tregs subsets from a cohort of transplanted patients under CNIs therapy and we investigate the effects of low-dose IL-2 therapy on Treg homeostasis and function in the presence of CNIs. Our data indicate that CNIs compromise the regulatory capacity of Tregs to a greater extent than previously reported by selectively promoting the apoptosis of the resting and activated Treg subsets, known to display the most powerful suppressive function. These effects are mainly caused by reduced access to IL-2, since Tregs remain capable to translocate nuclear NFAT even in the presence of high CNI levels. Exogenous IL-2 restores the phenotypic changes and overall gene expression effects exerted by CNIs and promotes Treg expansion by enhancing anti-apoptotic Bcl-2 expression. In a CNI-dependent transplant model, the addition of IL-2 resulted in the intra-graft accumulation of Tregs, reduced migration of effector T cells and prolonged allograft survival. Hence, combination of IL-2 and CNIs constitutes an optimal immunomodulatory regimen that enhances the pool of suppressive Treg subsets while effectively controlling cytopathic T cells.