2-Butylbenzo[d]isothiazol-3(2H)-one (BBIT, CAS No. 4299-07-4) is widely used as an industrial antiseptic and antifungal agent. To investigate its toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral toxicological study of BBIT was conducted in Crl:CD (SD) rats at doses of 0 (vehicle control, corn oil), 30, 90, or 270 mg/kg/day. There was no mortality or abnormal clinical signs related to treatment in any group. Slightly decreased body weight and food consumption were observed in the 270 mg/kg group in females. Increased urine volume and kidney weight, increased liver weight, and thickening of the forestomach mucosa in autopsy were observed in both sexes in the 270 mg/kg group. Histopathological examination revealed that hyperplasia of the squamous epithelium of the forestomach with parakeratosis and/or hyperkeratosis was observed in both sexes in all the BBIT-treated groups. Moreover, centrilobular hypertrophy of hepatocytes was observed in both sexes of the 270 mg/kg group. Similarly, increased depositions of eosinophilic bodies and/or hyaline droplets in the proximal tubules of the kidney were observed among the male in the 270 mg/kg group. Based on the forestomach changes, NOAEL was judged to be less than 30 mg/kg/day in both sexes under this study’s conditions.