To develop a stable mouse model of acute graft-versus-host disease (aGVHD) by preconditioning with busulfan (BS)-cyclophospha mide (CP).Male BALB/c (H-2kd) as recipients were conditioned with 100 mg/kg BS and 200 mg/kg CP followed by being transplanted with bone marrow cells (2×10(7)) or bone marrow cells (2×10(7))-spleen cells (2×10(7)) from female C57BL/6(H-2Kb) as donors. The general data of the transplanted mice were recorded and the manifestations of aGVHD were evaluated. Histopathological changes were observed using HE staining and chimerism in spleen and bone marrow cells was studied using flow cytometry.Mice transplanted with bone marrow cells-spleen cells developed classic aGVHD manifestations starting from the 7th day after transplantation with full donor chimerism. GVHD histopathological changes were seen in liver, spleen, intestine, skin and lung of these mice. The median survival time of these aGVHD mice was 10 days, and they all died within 30 days. Mice transplanted with only bone marrow cells survived over 45 days without any aGVHD signs.We developed a stable and reliable aGVHD murine model successfully, which can be used to study the pathogenesis, prevention and therapeutic effect of aGVHD.