Abstract Radiation‐induced intestinal injury is a serious concern during abdominal and pelvic cancers radiotherapy. Ubiquitin (Ub) is a highly conserved protein found in all eukaryotic cells. This study aims to explore the role and mechanism of free Ub against radiogenic intestinal injury. We found that free Ub levels of irradiated animals and human patients receiving radiotherapy were upregulated. Radiation‐induced Ub expression was associated with the activation of interferon regulatory factor 1 (IRF1). Intraperitoneal injection of free Ub significantly reduced the mortality of mice following 5–9 Gy total body irradiation (TBI) through the Akt pathway. Free Ub facilitates small intestinal regeneration induced by TBI or abdominal irradiation. At the cellular level, free Ub or its mutants significantly alleviated cell death and enhanced the survival of irradiated intestinal epithelial cells. The radioprotective role of free Ub depends on its receptor CXCR4. Mechanistically, free Ub increased fibroblast growth factor‐2 (FGF2) secretion and consequently activated FGFR1 signaling following radiation in vivo and in vivo. Thus, free Ub confers protection against radiation‐induced intestinal injury through CXCR4/Akt/FGF2 axis, which provides a novel therapeutic option.
