Objective To investigate the protective effect of puerarin on radiation-induced acute intestinal mucosal injury in rats and elucidate the underlying molecular mechanism. Methods Sprague-Dawley rats were randomly divided into three groups of eight rats each: a control group, radiation alone group, and radiation + puerarin group. The rats in the radiation alone group and radiation + puerarin group received a single dose of 12-Gy radiation, and the rats in the radiation + puerarin group were injected with 15 mg( kg·d) of puerarin via the tail vein for 7 consecutive days. Jejunal tissues were collected, and pathomorphologic changes in the intestine were observed by hematoxylin-eosin staining. The expression and distribution of Ki-67 antigen and platelet endothelial cell adhesion molecule 1 in jejunal tissues were detected by immunohistochemical staining. The protein expressions of claudin-1 and zonula occludens protein-1 (ZO-1) in jejunal tissues were detected by Western blot. The malonyldialdehyde level and superoxide dismutase activity in jejunal tissues were detected with the corresponding kits. Results Pathological examination showed that puerarin increased the villus length, maintained the villus epithelial structure, reduced the loss of villus epithelial cells, reduced the loss of crypts, promoted the regeneration of crypt cells, and reduced the density of goblet cells within crypts. Puerarin also significantly restored the reduced expression of claudin-1 and ZO-1 proteins and reduced the oxidative stress injury in jejunal tissues induced by radiation. Conclusions Puerarin protects rats from radiation-induced intestinal injury by promoting the expression of tight junction proteins, reducing oxidative stress injury, promoting the regeneration of crypt cells, and maintaining the integrity of the intestinal villus epithelial structure.