Low-dose interleukin-2 (IL-2) expands regulatory T cells (T_regs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of T_regs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m²/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of T_regs with increased suppressive function. There was a marked expansion of CD56^bright NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.