Isoeugenyl methyl ether (CAS No. 93-16-3) is a food additive used as a nature identical flavoring agent. To determine the toxicity profile and the no-observed-adverse-effect level (NOAEL), we performed a subchronic toxicity test in male and female F344/DuCrj rats by intragastric administration of isoeugenyl methyl ether at doses of 8, 40, and 200 mg/kg body weight (BW)/day for 13 weeks. In this study, BW gain in the male 200 mg/kg BW/day group was decreased from week 9. In serum biochemistry, decreased triglycerides were observed in the male 200 mg/kg BW/day group. In organ weights, increases in both absolute and relative liver weights were observed in both sexes in the 200 mg/kg BW/day group. In histopathological examination, hepatocyte hypertrophy was observed in the male 200 mg/kg BW/day group. Based on these results, we concluded that the main target organ of isoeugenyl methyl ether was the liver and that the NOAEL of isoeugenyl methyl ether for both male and female F344/DuCrj rats was estimated to be 40 mg/kg BW/day.