Abstract: Background　The intestine is one of the most radiosensitive organs. Currently available radioprotective agents are limited and often associated with high toxicity. Therefore, exploring whether glycerol, a low-toxicity compound, has protective effects against intestinal radiation injury is of great significance. Objective　To investigate the protective effect and underlying mechanism of glycerol against intestinal radiation injury and to evaluate its clinical potential.Methods　A mouse model of wholebody irradiation was established. Glycerol was administered at different doses and via different routes before irradiation to determine the optimal protocol based on jejunal crypt cell proliferation (BrdU staining) and stem cell number (Olfm4+ staining). The effects of glycerol on survival and body weight changes after 14 Gy irradiation were observed. Apoptosis in intestinal crypts after irradiation (Cleaved-Caspase 3, TUNEL) and the expression of related signaling pathways (P53-PUMA, γ -H2AX) were detected by immunohistochemistry and Western blot.Results　The optimal administration protocol was determined as intraperitoneal injection of 4 g/kg glycerol 3 h before irradiation, which significantly increased the number of BrdU+ proliferative crypts (43.1 ± 6.84 vs 19.1 ± 3.96, P ＜ 0.001). This regimen significantly prolonged the median survival time of irradiated mice (7.5 days vs 5.5 days, P ＜0.05) and mitigated body weight loss. Mechanistic studies revealed that glycerol significantly increased the number of Olfm4+ intestinal stem cells after irradiation (1.73 ± 0.22 vs 0.74 ± 0.23, P ＜ 0.001) and effectively suppressed crypt cell apoptosis at both 6 h (6.8 ± 1.32 vs 3.3 ± 1.16, P ＜ 0.001) and 24 h (4 ± 0.82 vs 2 ± 0.82, P ＜ 0.001) after irradiation. Furthermore, glycerol significantly inhibited the upregulation of P53 and PUMA proteins and reduced the level of the DNA damage marker γ-H2AX in intestinal tissue after irradiation. Conclusion　Glycerol, as a low-toxicity and safe compound, can mitigate radiation-induced intestinal crypt cell apoptosis and DNA damage by inhibiting the P53-PUMA signaling pathway, thereby protecting intestinal stem cells and prolonging survival. It demonstrates promising clinical potential for the prevention and treatment of radiation-induced intestinal injury.