To assess the toxicity of acenaphthylene, Sprague-Dawley rats were repeatedly administered the chemical via oral gavage at daily doses of 0, 4, 20, or 100 mg/kg/day for 28 days, followed by a 14-day recovery period. Decreases in body weight, food consumption, and body weight gain were observed in males and females in the 100 mg/kg/day group. Additionally, increases in water consumption and urine volume, and decreases in osmolality were observed in both males and females in this group. Moreover, this highest dose was linked to decreases in the reticulocyte percentage and increases in platelet counts in males and females, and females additionally exhibited increases in the hemoglobin concentration, mean corpuscular hemoglobin concentration, and activated partial thromboplastin time. Meanwhile, total cholesterol and phospholipid levels were elevated in males and females treated with 100 mg/kg/day acenaphthylene, with males additionally displaying increased total protein and albumin levels. Increased relative liver weights and changes in liver histopathology were observed in males and females treated with 20 or 100 mg/kg/day acenaphthylene. Additionally, organ weight and/or histopathological changes were observed in the thymus, heart, femoral and sternal bones including bone marrow, urinary bladder, kidneys, spleen, and adrenal gland in both sexes, in the stomach in males, and in the uterus, ovaries, and mesenteric lymph nodes in females in the 100 mg/kg/day group. Some changes exhibited plasticity in the recovery period. Based on these results, the no-observed-effect-level of acenaphthylene after repeated 28-day oral administration was 4 mg/kg/day.