Objective To establish a rat model of persistent intestinal mucosal injury. Methods The rat model of persistent intestinal mucosal injury was induced by intraperitoneal injection of methotrexate. The food intake and body weight of all the rats were recorded. Pathological changes were observed using HE staining, the level of D-lactate and diamine oxidase in plasma, and myeloperoxidase and malondiadehyde in the intestinal tissue were measured by biochemistry. Results After modeling, the rat body weight and food intake were decreased. On day 4, the scores of mucosal damage, the levels of plasma D-lactate, DAO, MPO and MDA were significantly increased (P<0.05). On day 5, the intestinal damages of rats began to restore, and there was no significant difference among groups on day 6. The symptoms after the secondary injection were similar to those after the first injection, and the rats recovered gradually at day 12. Conclusions Intestinal mucosal injury in rats induced by 20 mg/kg MTX is an acute injury process, the course only lasts for 4-5 days. Intermittent injections twice of 10 mg/kg MTX can cause persistent intestinal mucosal injury in rats. This persistent injury model is more suitable for nutritional therapy evaluation in medium-and long-term studies of nutritional therapy.