Background: Uncontrolled studies have suggested that bone marrow-derived mesenchymal stem cells (MSC) may be effective against acute graft-versus-host disease (aGvHD). We conducted a multicenter, randomized study to assess the efficacy, safety, and clinical benefit of using ex vivo cultured adult human MSC (remestemcel-L) in addition to second-line therapy to treat steroid refractory aGvHD (NCT00366145). Methods: Two-hundred sixty patients, 6 months to 70 years of age, were enrolled from August 2006 to May 2009, and were randomized 2:1 to receive 8 intravenous infusions of remestemcel-L or placebo, given over 4 weeks, in addition to second-line therapy according to institutional standards. Four additional infusions over 4 weeks were indicated for patients with incomplete response at Day 28. Randomization was stratified by aGVHD grade. Efficacy and safety were assessed through 180 days of follow-up, with the primary endpoint being durable complete response (DCR), defined as complete resolution of aGvHD symptoms for any period of at least 28 days after beginning treatment. Findings: Remestemcel-L did not meet the primary endpoint of greater DCR in the intent-to-treat population (35% vs. 30%; p=0.42). In post-hoc analyses patients with liver involvement who received at least one infusion, remestemcel-L had a higher DCR than those who received placebo (29% vs. 5%; p=0.047), and remestemcel-L produced favorable overall complete or partial response (OR) rates at day 28 compared to placebo among patients with liver involvement (55% vs. 26%; p=0.05), and among pediatric patients (64% vs. 23%; p=0.05). Infusions were well-tolerated and similar rates of adverse events were observed between treatment groups. Interpretation: Remestemcel-L was safe and well-tolerated. Results of this study did not demonstrate superior DCR compared to placebo when added to standard of care. The favorable clinical responses seen in some patient subsets may warrant further investigation. Funding: Mesoblast, Inc. (previous sponsor Osiris Therapeutics). Declaration of interest: AD, JU, DIM, EKW, and ES have nothing to disclose. RS has received consultancy fees from Gilead, Merck, and Astellas, served on board of directors for Kiadis, and DSMB of Juno/Celegene. PJM received consultancy fees from Enlivex Therapeutics, Genentech, and Grafalon, and served on DSMB for Pfizer. PK received consultancy fees from Jazz Pharmaceuticals. DS, EB and JH are employees of Mesoblast, Inc. Ethical Approval: The study protocol, all study protocol amendments, the written study patient information, written informed consent forms, and all other appropriate study-related information were reviewed and approved by an Independent ethics Committee (IEC) or Institutional Review Board (IRB) at each study site.