2-Ethylbutanal (2-EB) has been used as a flavoring agent. Here, we performed a 13-week subchronic toxicity study of 2-EB in F344 rats. 2-EB was given orally by gavage, using doses of 0, 50, 200 or 800 mg/kg BW/day. Reduced body weight gain was noted in both sexes at 800 mg/kg BW. Hematologic assessment showed a decrease in platelet counts in males at 200 mg/kg BW and both sexes at 800 mg/kg BW. Serum biochemistry demonstrated increases in inorganic phosphorus in both sexes at 200 and 800 mg/kg BW, increases in glucose in females at 200 and 800 mg/kg BW and increases in urea nitrogen in both sexes at 800 mg/kg BW. Regarding organ weights, increases in absolute and relative weights of the liver and kidney with toxicological significance were detected in both sexes at 200 and 800 mg/kg BW. Hepatocellular hypertrophy with eosinophilic granular cytoplasmic changes in the liver were observed in males at 200 mg/kg BW and in both sexes at 800 mg/kg BW. Necrosis/regeneration of proximal tubules in the kidney was detected in females at 800 mg/kg BW. Based on these results, the no-observed-adverse-effect level (NOAEL) of 2-EB was evaluated to be 50 mg/kg BW/day for both sexes.