Low-dose methotrexate (LD-MTX) has long been included in prophylaxis regimens for acute graft-versus-host disease (aGVHD). In addition, LD-MTX is expected as a treatment option to control aGVHD by regulating the cytokine network. In this study, we retrospectively evaluated 35 patients with steroid-refractory acute GVHD to evaluate the safety and efficacy of LD-MTX as a salvage treatment. LD-MTX was administered weekly at a dose of 10 mg/m(2). Overall, 13 patients (37 %) achieved complete response and three (9 %) achieved partial response within 4 weeks after LD-MTX was initiated without any additional agents. Resolution of manifestations of aGVHD in each evaluable organ was observed in 12 of the 23 cases (52 %) with skin aGVHD and in eight of the 23 cases (35 %) with GI aGVHD. Neutropenia and thrombocytopenia (grades III and IV) were observed in nine (26 %) and 17 patients (49 %), respectively. Fatal infectious complications occurred in only three patients (9 %) after LD-MTX treatment. Of the 35 patients studied, 22 were alive with a median follow-up of 60 months and an overall survival rate (Kaplan-Meier estimate) was 62 %. LD-MTX seems suitable for salvage therapy and will not increase risk of infection. Further evaluation of the use of LD-MTX as salvage therapy for steroid-refractory acute GVHD is warranted.