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Acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT).Our previous study found that the novel anti-inflammatory cytokine IL-35 could suppress aGVHD in patients after allo-HSCT.In this study, we used C57BL/6 (B6, H-2b) mice as donors and (B6 × DBA/2)F1 (BDF1, H-2b × d) mice as recipients to create a model of aGVHD and explore the relationship between IL-35 and aGVHD.The mice receiving IL-35 survived longer than did the control mice.We observed that treatment with IL-35 and RAPA could reduce the incidence of aGVHD.Additionally, this treatment inhibited intestinal and thymic epithelial cell apoptosis and liver infiltration by the donor T-cells, thereby ameliorating the enteropathy and liver injury caused by aGVHD.We found that IL-35 and RAPA also markedly suppressed TNF-α and IL-17A expression and enhanced IFN-γ expression in the intestine and liver.We measured Tregs in spleen and found that IL-35 and RAPA treatment expanded the number of Tregs in spleen.We found that the phosphorylation of STAT1 and STAT4 were inhibited in mice with aGVHD.In contrast, STAT1 and STAT4 were phosphorylated when the mice were treated with IL-35.IL-35 may have therapeutic potential in the treatment of aGVHD after allo-HSCT.