<p>Pyroptosis, a novel form of inflammation-related programmed cell death that often occurs in myeloid cells, plays an important role in multiple inflammatory diseases. Our previous study revealed macrophage dysfunction in acute graft-versus-host disease (aGVHD). However, whether macrophages undergo pyroptosis in aGVHD remains unknown. In our study, macrophage pyroptosis was observed in aGVHD mice. Additionally, serum IL-1β and IL-18 levels were increased in aGVHD patients. Almost all peritoneal macrophages in the aGVHD mice were derived from the donors. Less pathological damage and a longer survival time were observed in the mice that received purified T cells and bone marrow (BM) from AAV9-F4/80-GSDMD mice, indicating that macrophage pyroptosis in donor mice promoted the development of aGVHD. In addition, decreased proportions of CD69⁺CD4⁺ T cells, Th1 cells and Th17 cells and an increased proportion of Tregs were observed in the recipients when GSDMD was suppressed in the donor macrophages. Administration of a pyroptosis inhibitor significantly alleviated the severity of aGVHD without impairing graft-versus-lymphoma (GVL) effects. Our results suggested that donor-derived macrophages undergo pyroptosis in aGVHD, and these cells might participate in the development of aGVHD by affecting the activation and differentiation of CD4⁺ cells. The pyroptosis inhibitor disulfiram is a potentially promising agent for aGVHD treatment.</p>