Background/Aim: This study aimed to develop a reliable chemotherapy-induced oral mucositis (CIOM) rat model by intraperitoneally administering a single dosage of 5-fluorouracil (5-FU) combined with a chemical stimulus. Materials and Methods: The 5-FU dosage for CIOM development was determined by the survival rate of rats administrated 160 mg/kg, 200 mg/kg, and 240 mg/kg of 5-FU. Thirty rats were assigned to normal control (NC) and three experimental groups: i) ulcer formation without 5-FU administration (PBS/U+), ii) 5-FU administration without ulcer formation (5-FU/U-), and iii) ulcer formation after 5-FU administration (5-FU/U+). White blood cell count and weight were measured at the day of 5-FU administration (D0), ulcer formation (D2), and two days after ulcer formation (D4). The oral mucosa for histologic evaluations was obtained two (D4) and five days (D7) after ulcer formation. Results: The 5-FU dosage for CIOM development was 200 mg/kg. White blood cell count (WBC) counts and weight of rats were significantly lower in 5-FU/U- (WBC, p<0.001; weight, p=0.002) and 5-FU/U+ (WBC, p<0.001; weight, p<0.001) groups compared to those in the NC group at D4. The number of Ki-67 positive cells in the oral epithelium was lower in 5-FU/U+ group compared to that in NC (p<0.001) and PBS/U+ (p=0.047) groups at D7. Conclusion: Single administration of 200 mg/kg of 5-FU combined with a chemical stimulus can lead to an immunesuppressive status, failure of weight gain, and impairment of epithelium regeneration as observed in a CIOM rat model.