GVHD is still one of the major complications after allogeneic stem cell transplantation. Whereas murine data have clearly shown the beneficial effects of regulatory T cells (Tregs) on the prevention of GVHD, data from the human system are rare. Here, we present a comparative dynamic analysis of CD4(+)CD25(hi)CD127(lo/-) Tregs from patients with and without GVHD analyzing the whole genome profile over the first 6 months after stem cell transplantation, representing the most sensitive time window for tolerance induction. The Treg transcriptome showed a high stability. However, the comparison of Treg transcriptomes from patients with and without GVHD uncovered regulated gene transcripts highly relevant for Treg cell function. The confirmative protein analyses demonstrated a significantly higher expression of granzyme A, CXCR3, and CCR5 in Tregs of immune tolerant patients. These results point to a reduced suppressive function of Tregs from GVHD patients with diminished migration capacity to the target organs.