Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT), and damage to the gastrointestinal (GI) tract plays a critical role in amplifying systemic disease. Intestinal stem cells (ISCs) play a pivotal role not only in physiological tissue renewal but also in regeneration of the intestinal epithelium after injury. In this study, we have discovered that pretransplant conditioning regimen damaged ISCs; however, the ISCs rapidly recovered and restored the normal architecture of the intestine. ISCs are targets of GVHD, and this process of ISC recovery was markedly inhibited with the development of GVHD. Injection of Wnt agonist R-spondin1 (R-Spo1) protected against ISC damage, enhanced restoration of injured intestinal epithelium, and inhibited subsequent inflammatory cytokine cascades. R-Spo1 ameliorated systemic GVHD after allogeneic BMT by a mechanism dependent on repair of conditioning-induced GI tract injury. Our results demonstrate for the first time that ISC damage plays a central role in amplifying systemic GVHD; therefore, we propose ISC protection by R-Spo1 as a novel strategy to improve the outcome of allogeneic BMT.