The total flavonoids (TFs) from Rosa laevigata Michx fruit showed hepatoprotective and antioxidant activities. However, the safety of this natural product has not been investigated. In the present paper, a 90-day subchronic toxicity study was conducted, and the tested TFs was orally administered to rats at the doses of 500, 1000 and 2000 mg/kg/day. The toxicity of the TFs was evaluated on base of ophthalmic examination, body weight, feed/water consumption, urinalysis, hematology, clinical biochemistry and pathology. No toxic signs of the TFs at the doses of 500 and 1000 mg/kg/day were observed. However, decreased PLT was found in the 2000 mg/kg/day groups and increased intercellular space of myocardial cells was observed in the male 2000 mg/kg/day group compared with control. A significant increase in the relative cardiac weight was observed in the male 1000 and 2000 mg/kg/day groups. And the significant decrease in the absolute and relative weight of adrenals in the female 1000 and 2000 mg/kg groups was happened. The TFs could cause mild side effects at the dose of 1000 mg/kg/day in males and females. Thus, the dose of 500 mg/kg/day for male and female were selected as the no-observed-adverse-effect level (NOAEL). The present study provides useful data for subsequent researches and new drug exploration of the TFs from R. laevigata Michx fruit.