Acute graft-versus-host disease (aGVHD) is a major, life-threatening complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the development of novel molecular-targeted therapies for aGVHD is urgently needed. Thiolutin (THL), a disulfide-containing antibiotic, inhibits the activity of the BRCC3-containing isopeptidase complex and thereby suppresses NLR pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Here, we investigated the effect of THL on aGVHD in a mouse model of allo-HSCT. We found that THL significantly improved survival in aGVHD recipients, reduced weight loss and clinical scores, and attenuated pathological changes in aGVHD target organs, including the liver, small intestine, and colon. Furthermore, THL diminished donor T-cell activation and infiltration into target organs and promoted the expansion of regulatory T cells. THL also inhibited NLRP3 inflammasome activation and reduced the production of proinflammatory cytokines, including tumor necrosis factor (TNF), interferon γ (IFN-γ), interleukin (IL)-2, IL-17A, and IL-1β. This study reveals a previously unrecognized role of THL in regulating aGVHD and highlights its potential as an anti-inflammatory therapeutic agent for clinical development in patients undergoing allo-HSCT.