Background <p>Dysbiosis of the intestinal microbiota plays a crucial role in the initiation and development of graft-versus-host disease (GVHD). Fecal microbiota transplantation (FMT) has been reported to be effective for refractory acute GVHD; however, whether FMT is effective for refractory chronic GVHD (cGVHD) remains unknown.</p> Methods <p>To investigate the efficacy and safety of FMT for refractory cGVHD and the underlying mechanism, 12 patients with refractory cGVHD received FMT via colonoscopy, and the response was evaluated at 12&#xa0;weeks after FMT.</p> Results <p>Among the 12 patients who underwent FMT, 1 patient achieved a complete response, and 5 patients achieved a partial response. Patients with refractory cGVHD presented lower α diversity and higher abundance of <i>Escherichia-Shigella </i>and<i>Enterobacteriaceae</i>. FMT increased gut microbial diversity, increased the abundance of short-chain fatty acid (SCFA)-producing bacteria, and decreased the abundance of <i>Escherichia-Shigella</i> and <i>Enterobacteriaceae</i> in responder patients. Moreover, it increased SCFA levels in fecal samples from the responder group and promoted the expansion of peripheral CD4⁺CD127^- regulatory T (Treg) cells. Colon pathological examination revealed that CD4⁺ T and CD19⁺ B cell infiltration decreased and that CD4⁺ Treg infiltration increased after FMT.</p> Conclusions <p>The results of the present study suggest that FMT is feasible and deserves further investigation for use in patients with refractory cGVHD.</p> Trial registration <p>ClinicalTrials.gov (NCT06938165).</p>