Sirolimus is an inhibitor of the mammalian target of rapamycin (mTOR) and is emerging as a promising component of graft-versus-host disease (GVHD) prophylaxis regimens in the context of allogeneic hematopoietic stem cell transplantation (HSCT). Multiple studies have explored the clinical benefits of adding sirolimus to GVHD prophylaxis, however detailed immunological studies have not yet been carried out in this context. Mechanistically, mTOR is at the center of metabolic regulation in T- and NK cells and is critical for their differentiation to mature effector cells. Therefore, close evaluation of the inhibition of mTOR in the context of immune reconstitution post HSCT is warranted. To study the effect of sirolimus on the immune reconstitution using a biobank of longitudinal samples from patients receiving either tacrolimus/sirolimus (TAC/SIR) compared to cyclosporin A/methotrexate (CSA/MTX) as conventional GVHD prophylaxis. Healthy donor controls, donor graft material, and samples from 28 patients (14 TAC/SIR, 14 CSA/MTX) at 3-4 weeks and 34-39 weeks post HSCT were collected. Multi-color flow cytometry was used to carry out a broad immune cell mapping with a particular focus on NK cells. NK cell proliferation was evaluated over a 6-day in vitro homeostatic proliferation protocol. Furthermore, in vitro NK cell responses to cytokine stimulation or tumor cells were evaluated. Systems-level assessment of the immune repertoire revealed a deep and prolonged suppression (week 34-39 after HSCT) of the naïve CD4 T cell compartment with a relative sparing of regulatory T cells and an enrichment of CD69+Ki-67+HLA-DR+ CD8 T cells, independent of the type of GVHD prophylaxis. Early after transplantation (week 3-4), while patients were still on TAC/SIR or CSA/MTX, we found a relative increase in less differentiated CD56bright NK cells and NKG2A+CD57-KIR- CD56dim NK cells and a distinct loss of CD16 and DNAM-1 expression. Both regimens led to suppressed proliferative responses ex vivo and functional impairment with preferential loss of cytokine responsiveness and IFN-γ production. Patients that received TAC/SIR as GVHD prophylaxis showed delayed NK cell reconstitution with lower overall NK cell counts, fewer CD56brights and NKG2A+ CD56dim NK cells. Treatment with Sirolimus containing regimens generated similar immune cell profiles as conventional prophylaxis. However, the NK cell compartment seemed to be composed of slightly more mature NK cells. These effects were also present after GVHD prophylaxis end, suggesting that mTOR-inhibition with sirolimus leaves a lasting imprint on homeostatic proliferation and NK cell reconstitution following HSCT.