Amifostine (AM) is a radioprotector that scavenges free radicals and is used in patients undergoing radiotherapy. p53 has long been implicated in cell cycle arrest for cellular repair after radiation exposure. We therefore investigated the protective p53-dependent mechanism of AM on small bowel damage after lethal whole-abdominal irradiation (WAI). AM increased both the survival rate of rats and crypt survival following lethal 18 Gy WAI. The p53 inhibitor PFT-α compromised AM-mediated effects when administered prior to AM administration. AM significantly increased clonogenic survival in IEC-6 cells expressing wild type p53 but not in p53 knockdown cells. AM significantly increased p53 nuclear accumulation and p53 tetramer expression before irradiation through the inhibition of p53 degradation. AM inhibited p53 interactions with MDM2 but enhanced p53 interactions with 14-3-3σ. Knockdown of 14-3-3σ also compromised the effect of AM on clonogenic survival and p53 nuclear accumulation in IEC-6 cells. For the first time, our data reveal that AM alleviates lethal small bowel damage through the induction of 14-3-3σ and subsequent accumulation of p53. Enhancement of the p53/14-3-3σ interaction results in p53 tetramerization in the nucleus that rescues lethal small bowel damage.