The aim of the study was to evaluate the safety of flavonoid fraction of Lithocarpus polystachyus Rehd (Sweet Tea-F, ST-F) in mice and rats through acute and sub-chronic toxicity studies respectively. For acute toxicity study, a single dose of 5000 mg/kg of ST-F was given orally to healthy KM mice. The mice were observed mortality and toxic symptoms for 24 h, then once a day up to 14 days. In the sub-chronic toxicity study, ST-F was administered orally at doses of 0, 70, 140, 560 mg/kg/day to rats for 26 weeks. Body weight and food intake were recorded weekly. Hematological, biochemical, coagulation and organ parameters were analyzed at the end of 26 weeks administration. Vital organs were evaluated by histopathology. In the acute toxicity study, ST-F caused neither significant toxic symptoms, nor mortality in mice. In sub-chronic toxicity study, daily oral administration of ST-F at the dose of 70 mg/kg resulted in a significant increase (P < 0.05) in the relative body weight at the 10-week, and the same situation brought at the dose of 140 mg/kg/day at the 22-week. Hematological and biochemical showed significant changes (P < 0.01 or P < 0.05) in WBC, GLU, ALP, AST and serum electrolytes levels at the dose of 560 mg/kg/day. The amount of RBC decreased significantly (P < 0.05) while the content of PLT slightly increased (P < 0.05) at the dose of 140 mg/kg/day. In additional, no obvious histological changes were observed in vital organs of ST-F treated animals compared to control group. The ST-F may be exit slight side effects at the dose of 560 mg/kg/day in rats. Thus, the overall results show that the no-observed adverse effect level (NOAEL) of ST-F was considered to be 140 mg/kg for male SD rats.