Severe graft-versus-host disease (GVHD) is a lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, there have been only a few limited therapeutic options for the treatment of GVHD. Here we have reported two cases of the patients with acute steroid-refractory GVHD who underwent allogeneic HSCT. These patients received infusions of third-party clonal mesenchymal stem cells (cMSCs), which were isolated by a subfractionation culturing method (SCM) developed recently by our group, and showed marked improvement of the disease. MSCs represent a new potential therapeutic option in the treatment of steroid-refractory acute GVHD. They showed safety and improved clinical findings of the acute GVHD patients. However, further investigations are needed to understand the accurate mechanisms of MSCs and larger well-designed human clinical trials are necessary to prove further the safety and efficacy of the cMSCs for the treatment of acute GVHD patients.