This study aimed to explore macrophage polarization in acute graft-versus-host disease after hematopoietic stem cell transplantation, and investigated if arsenic trioxide (ATO) could correct this imbalance. In the colon of GVHD mice, we found that the number of F4/80⁺iNOS⁺ cells as well as the expression intensity of TNF-α and IL-1β was greater in the GVHD group than in the BM group, whereas the number of F4/80⁺CD206⁺ cells and the expression intensity of IL-10 and TGF-β was greater in the BM group than in the GVHD group. We investigated the effect of ATO on GVHD mice, and found that ATO treatment clearly improved the survival of the mice and reduced the severity of GVHD. In addition, ATO reduced the number of F4/80⁺iNOS⁺ cells, and increased the number of F4/80⁺CD206⁺ cells in the colon of GVHD mice. Furthermore, ATO sharply decreased CD86 and CD80 expression, and increased CD163 and CD206 expression in macrophages induced from aGVHD patients. Therefore, ATO can modulate the M1 and M2 phenotype in GVHD mice or in macrophages from aGVHD patients. Our data suggest that macrophage polarization is involved in the pathogenesis of aGVHD, and ATO treatment modulates macrophage polarization toward an M2 phenotype.