Macrophages (MΦs) are an important immune cell population that are essential for tissue homeostasis and disease pathogenesis. MΦs are now classified as either M1, which produce pro-inflammatory cytokines, or M2, which produce antiinflammatory cytokines. The impact of granulocyte colony-stimulating factor (G-CSF) on MΦs in humans is unclear. Moreover, little is known about the association between MΦ subsets in allografts and the occurrence of acute graft-versus-host disease (aGVHD) in patients who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the current study, we found that the M1/M2 ratio was markedly decreased in both G-CSF-treated bone marrow (post-BM) and G-CSF-treated peripheral blood from healthy donors. Post-BM MΦs exhibited reduced migration and increased phagocytosis. Moreover, post-BM MΦs reduced the percentage of Th1 and Tc1 lineages and increased the percentage of Th2, Tc2, and Treg lineages. Patients who received BM grafts with a higher M1/M2 ratio exhibited a higher incidence of grade 2-4 aGVHD. In summary, our data indicate that G-CSF decreases the M1/M2 ratio in BM grafts from healthy donors, which may contribute to preventing the occurrence of grade 2-4 aGVHD in patients after allo-HSCT.