Acute graft-versus-host disease (GVHD) is characterized by severe tissue damage that is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been increasingly examined for the treatment of immune-related diseases. We aimed to assess the immunosuppressive effects of human amnion-derived MSC (AMSC) in a xenogeneic GVHD NOD/Shi-scid IL2rγnull mouse model using human peripheral blood mononuclear cells (PBMC). Additionally, we used human bone marrow-derived MSC (BMSC) as comparative controls to determine differences in immunomodulatory functions depending on the MSC origin. Administration of AMSC significantly prolonged survival, and reduced human tumor necrosis factor-α (TNF-α) concentration and percentage of programmed cell death protein-1 receptor (PD-1)+CD8+ T cell populations compared with in GVHD control mice. Furthermore, colonic inflammation score and percentage of human CD8+ T cell populations in AMSC-treated mice were significantly lower than in GVHD control and BMSC-treated mice. Interestingly, gene expression and protein secretion of the PD-1 ligands were higher in AMSC than in BMSC. These findings are the first to demonstrate that AMSC exhibit marked immunosuppression and delay acute GVHD progression by preventing T cell activation and proliferation via the PD-1 pathway.