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Background Mesenchymal stem cells (MSCs) may be used to treat steroid-refractory graft versus host disease (GVHD). However, the effects of MSCs in haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) have not been confirmed in randomized studies. Methods We conducted a randomized clinical study to investigate the effects of pre-infusion (1 × 10 6 cells/kg) MSCs on hematopoietic recovery, Epstein–Barr and cytomegalovirus infection, GVHD, and relapse in patients undergoing haplo-PBSCT. Fifty patients with acute leukemia or myelodysplastic syndrome were randomly divided into an MSC group administered 1 × 10 6 MSCs/kg 4 to 6 hours before infusion of peripheral stem cells and a control group without MSCs. Results Mean platelet engraftment time was significantly faster in the MSC compared with the control group (12.28 vs 13.29 days). The mean neutrophil engraftment time was comparable in both groups (10.76 ± 2.40 vs. 10.29 ± 1.72 days). Grade II or above acute GVHD was significantly decreased in the MSC compared with the control group (12% vs. 36%). There were no significant differences in relapse rate or overall survival between the groups. Conclusion These results suggest that pre-infusion single-dose MSCs promote platelet engraftment and decrease severe acute GVHD without increasing relapse rate.