2,4-Dimethyl-4-phenyltetrahydrofuran (CAS no. 82461-14-1) is a food additive used as a synthetic flavoring substance. To investigate the toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral dose toxicity study of 2,4-dimethyl-4-phenyltetrahydrofuran containing four stereoisomers was conducted in F344 rats at doses of 0, 6, 24, and 96 mg/kg body weight (BW)/day. No mortality or abnormal clinical signs related to treatment in any group was observed. At a dose of 96 mg/kg BW, fluctuated serum total protein and total cholesterol and increased absolute and relative liver weights and relative kidney weights were observed in both sexes. Increased serum albumin in males and decreased Na and Cl in females were also observed. On histopathological assessment, at a dose of 96 mg/kg BW, diffuse hepatocellular hypertrophy in the liver in both sexes and tubular regeneration with scattered proximal tubular degeneration and/or necrosis throughout the cortex in the kidney in males were detected. Based on these findings, the NOAEL for 2,4-dimethyl-4-phenyltetrahydrofuran used in the current study was found to be 24 mg/kg BW/day for both sexes.