A 28-day repeated-dose oral toxicity test was performed to determine the no-observed-effect level (NOEL) and establish an optimum dose of the highly toxic Aconiti Ciliare Tuber (ACT) used as a folk remedy. Repeated oral doses of 1,250, 2,500, and 5,000 mg/kg/day of the hot water extract of ACT were administered to five male and five female Sprague-Dawley rats in each group for 4 weeks. The indicators for toxicity included results of examination of common symptoms and changes in weight and feed intake, eye test, urinalysis, hematological and serum biochemical analyses, and post-mortem weight measurement of organs, and visual inspections. All animals survived at the end of the experiment; in addition, we observed no specific test substance-mediated symptoms. We observed no test substance-mediated changes in body weight and feed intake. We observed statistically significant changes in male OB and pH levels (p<0.05). Further, the biochemical test showed statistically significant changes in the IP value of male rats and $CL^-$valueoffemalerats (p<0.05). However, all changes were within historical data. The post-mortem examinations showed no test substance-mediated changes. Moreover, statistically significant changes under the test conditions were confirmed to have been caused by factors other than the test substance. Thus, the maximum NOEL of ACT extract in rats was estimated to be 5,000 mg/kg/day.