Objective To establish a mouse model of 5-fluorouracil ( 5-FU )-induced intestinal mucositis. Methods Different doses of 5-FU were intraperitonealy injected into mice for once or 5 consecutive days. The body weight and diarrhea score of the mice were recorded every day, and peripheral blood and the morphological changes of small intestine were examined at 24 h or 72 h after the last 5-FU administration. Results Compared with the control group, after administered 5-FU, the dosage groups showed various degrees of body weight loss and diarrhea, and the white blood cell and platelet counts in peripheral blood decreased significantly (P＜0. 05 or P＜0. 01). The small intestine showed evident pathological changes after the single dose 400 mg/kg and the continuous injection of 50 mg/kg, 100 mg/kg. The mortality rate was 100% in the 100 mg/kg group. Conclusions A mouse model of intestinal mucositis can be successfully established by a single injection of 5-FU or for consecutive 5 days, in a dose-dependent manner. The optimal dose for single injection is 400 mg/kg, and that of the continual injection for 5 consecutive days is 50 mg/kg.