Konjac mannan oligosaccharide (KMOS) is a new food additive with prebiotic function in China. In this study, KMOS with degree of polymerization (DP) of 2～7 was prepared by enzymatic method followed by organic solvent precipitation. Then its subchronic toxicity and genotoxicity was investigated. In the subchronic toxicity test, KMOS was administered to rat orally for 90 days at dose of 0, 2.25, 5.25, 7.50 g/kg body weight (BW) daily, respectively. No significant toxicological manifestation in clinical examination as well as clinical pathology was noted. At terminal necropsy, histopathology changes in the liver and kidney were observed, which was considered to be spontaneous and incidental in nature and unrelated to KMOS-treatment. In addition, a battery of tests including the Ames test, micronucleus test and sperm abnormality test suggested no mutagenicity potential. In conclusion, the results of this study supported that ingestion of KMOS appeared to be safe as a food additive for oral consumption.