Posttransplant high-dose cyclophosphamide (PTCY) has been increasingly used as graft-versus-host disease (GVHD) prophylaxis after HLA-haploidentical or matched hematopoietic stem cell transplantation (SCT). However, PTCY alone is insufficient and requires additional immunosuppressants such as calcineurin inhibitors. In the current study, we evaluated effects of a novel GVHD prophylaxis with PTCY in combination with short-term KRP203, a selective agonist of sphingosine-1-phosphate receptor 1 that regulates egress of lymphocytes from the secondary lymphoid organs (SLOs) in mice. Short-term oral administration of KRP203 alone induced apoptosis of donor T cells in the SLOs and ameliorated GVHD. Administration of KRP203 significantly preserved graft-versus-leukemia effects compared to cyclosporin. A combination of KRP203 on days 0 to +4 and PTCY on day +3 synergistically suppressed donor T-cell migration into the intestine and skin, and ameliorated GVHD more potently than PTCY alone. A combination of short-term KRP203 and PTCY is a promising novel calcineurin-free GVHD prophylaxis in HLA-haploidentical SCT.