Cynanchi wilfordii Radix (CWR) is a herbal medicinal plant that is well-known and used in Asian countries as a health food. In this study, acute and 13-week subchronic oral toxicity studies of hot-water extract of CWR (CWR-WE) were performed in Sprague-Dawley rats. For the acute toxicity study, CWR-WE was administered once orally to five male and five female rats at doses of 800, 2000, and 5000 mg/kg. Mortality, clinical signs, and body weight changes were monitored over 14 days. There were no treatment-related changes in these parameters and the approximate lethal dose of CWR-WE in male and female rats was determined to be > 5000 mg/kg. For the subchronic toxicity study, CWR-WE was administered orally once daily to male and female rats for 13 consecutive weeks at doses of 0 (vehicle control), 500, 1000, and 2000 mg/kg/day (n = 10 rats/sex/group). There were no toxicologically significant changes with regard to clinical signs, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, organ weights, necropsy findings, and histopathological findings. These results suggest that the oral no observed adverse-effect level of CWR-WE is > 2000 mg/kg/day for both sexes, although target organs were not identified.