Intestinal microbiome diversity plays an important role in the pathophysiology of acute gastrointestinal (GI) graft-versus-host disease (GVHD) and influences the outcome of patients after allogeneic stem cell transplantation (ASCT). We analyzed clinical data and blood samples taken preconditioning and on the day of ASCT from 587 patients from 7 German centers of the Mount Sinai Acute GVHD International Consortium, dividing them into single-center test (n = 371) and multicenter validation (n = 216) cohorts. Regenerating islet-derived 3 alpha (Reg3 alpha) serum concentration of day 0 correlated with clinical data as well as urinary 3-indoxylsulfate (3-IS) and Clostridiales group XIVa, indicators of intestinal microbiome diversity. High Reg3 alpha concentration at day 0 of ASCT was associated with higher 1-year transplant-related mortality (TRM) in both cohorts (P < .001). Cox regression analysis revealed high Reg3 alpha at day 0 as an independent prognostic factor for 1-year TRM. Multivariable analysis showed an independent correlation of high Reg3 alpha concentrations at day 0 with early systemic antibiotic (AB) treatment. Urinary 3-IS (P = .04) and Clostridiales group XIVa (P = .004) were lower in patients with high vs those with low day 0 Reg3 alpha concentrations. In contrast, Reg3 alpha concentrations before conditioning therapy correlated neither with TRM nor disease or treatment-related parameters. Reg3 alpha, a known biomarker of acute GI GVHD correlates with intestinal dysbiosis, induced by early AB treatment in the period of pretransplant conditioning. Serum concentrations of Reg3 alpha measured on the day of graft infusion are predictive of the risk for TRM of ASCT recipients.