This trial evaluated the feasibility and efficacy of the infusion of mesenchymal stem cells expanded using human serum for the treatment of refractory acute or chronic graft-versus-host disease. Twenty-eight expansions were started. In 22, a minimum of more than 1 x 10⁶ mesenchymal stem cells/kg were obtained after a median of 26 days; this threshold was not obtained in the remaining cases. Ten patients received cells for the treatment of refractory or relapsed acute graft-versus-host disease and 8 for chronic disease. One patient treated for acute graft-versus-host disease obtained a complete response, 6 had a partial response and 3 did not respond. One of the chronic patients achieved complete remision, 3 a partial response, and 4 did not respond. The current study supports the use of this approach in less heavily treated patients for both acute and chronic graft-versus-host disease. The trial has been registered at ClinicalTrials.gov: identifier NCT00447460.