Objective To investigate the molecular mechanism by which fecal microbiota transplantation (FMT) alleviates gastrointestinal inflammation after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in murine acute graft-versus-host disease (aGVHD). Methods A murine aGVHD model after allo-HSCT was established, and BALB/c mice were randomly assigned to blank control, bone marrow transplantation, aGVHD model, and FMT treatment groups (n=6 per group). Disease severity was assessed by histopathology. Expression of receptor-interacting protein kinase (RIPK)1, RIPK3, and mixed lineage kinase domain-like protein (MLKL) was evaluated by immunohistochemistry. Protein levels of RIPK1, RIPK3, MLKL, phosphorylated RIPK1 (p-RIPK1), and phosphorylated MLKL (p-MLKL) were determined by Western blotting. Plasma regenerating islet-derived protein 3 alpha (Reg3α) was measured by enzyme-linked immunosorbent assay. The intestinal microbiota was profiled by 16S rRNA gene sequencing. Results Compared with the aGVHD model group, the FMT group showed higher relative abundances of Firmicutes and Bacteroidetes and a lower relative abundance of Proteobacteria; body weight loss was markedly attenuated, and survival time was prolonged. Alpha-diversity indices (Simpson, Pielou, Shannon) increased in the FMT group (P<0.05). Intestinal pathology scores, expression of RIPK1, RIPK3, and MLKL, protein levels of RIPK1, RIPK3, MLKL, p-RIPK1, and p-MLKL, and plasma Reg3α levels were significantly reduced in the FMT group versus the aGVHD model group (all P<0.05). Conclusions FMT may attenuate gastrointestinal inflammation in aGVHD by restoring intestinal microbial balance and inhibiting the RIPK1/RIPK3-mediated necroptosis pathway.