The object of this study was two. One was if Polygonati Rhizoma preparata had a benzo(a)pyrene, the other was to evaluate the single dose toxicity of 9 repetitive steaming and fermenting processed Polygonati Rhizoma, dried root parts of Polygonati Rhozoma preparata extract, in male and female mice. We measured a content of benzo(a)pyrene in Polygonati Rhozoma preparata using a method with HPLC/FLD. And for single dose toxicity, aqueous extracts of Polygonati Rhozoma preparata (EPP; Yield = 35.4 %) was administered to female and male ICR mice as an oral dose of 2,000, 1,000 and 500 mg/kg (body weight) according to the recommendation of Korea Food and Drug Administration (KFDA) Guidelines. Animals were monitored for the mortality and changes in body weight, clinical signs and gross observation during 14 days after dosing, upon necropsy; organ weight and histopathology of 12 principle organs were examined. As results, we could not find any mortality, clinical signs, and changes in the body and organ weight except for slight soft feces sporadically detected in EPP treated male mice at 1 day after administration. In addition, no EPP-treatment related abnormal gross findings and changes in histopathology of principle organs were detected except for some sporadic accidental findings. The results obtained in this study suggest that benzo(a)pyrene was not existed in Polygonati Rhozoma preparata and the 50% lethal dose and approximate lethal dose of EPP aqueous extracts in both female and male mice were considered as over 2,000 mg/kg, the limited highest dosage recommended by KFDA Guidelines. However, it also observed that the possibilities of digestive disorders, like soft feces when administered over 500 mg/kg of EPP aqueous extracts in the present study.