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Severe steroid-resistant acute graft-versus-host disease (aGVHD) is associated with high mortality. Bone marrow-derived mesenchymal stem cells (BMMSC) have been found to be immunosuppressive, and intravenous infusion of BMMSC is an effective therapy for steroid-resistant aGVHD. However, acquiring BMMSC requires an invasive procedure.We compared umbilical cord-derived mesenchymal stem cells (UCMSC) and BMMSC for morphology, surface markers expression, differentiation, proliferative potential, and their suppressive effects on peripheral blood mononuclear cell proliferation. After institutional review board approved, we intravenously infused ex vivo expanded third-party UCMSC into two patients with severe steroid-resistant aGVHD. Adverse effects and patient responses of UCMSC were monitored. All procedures for UCMSC processing complied with current good tissue practice requirements.We found that UCMSC had superior proliferative potential and more suppressive effects on peripheral blood mononuclear cell proliferation compared with BMMSC. The aGVHD improved dramatically after each of four infusions of UCMSC into the two patients. No adverse effects were noted. Both patients are doing well now.Considering that acquiring UCMSC is noninvasive, these cells would appear to be the ideal candidates for clinical cell-based therapies. This is the first report of UCMSC in a human clinical application, and this procedure seems both feasible and safe. These findings suggested that UCMSC were effective for treating aGVHD.