Abstract: Response surface methodology（RSM）was used to optimize the extraction conditions of guaijaverinand avicularin from guava leaves based on single-factor tests. The best harvest month was valued according to thecontent of guaijaverin and avicularin from guava leaves. Besides，the hypoglycemic activity in vitro and the acutetoxicity of guava leaves harvested in the best month was evaluated by the isolated rat adipocyte model and miceseparately. Results showed that the optimum extraction conditions by RSM were as follows：extraction time of 81 min，ratio of liquid to material of 11∶1，ethanol concentration of 64.6%. Under these conditions，the yield of guaijaverin andavicularin was 3.40（±0.02）mg/g. The repeated tests showed that the optimal conditions were stable and feasible. Thetotal content of guaijaverin and avicularin was different in guava leaves from different months. The highest contentof guaijaverin and avicularin was from July，which wasup to 6.74（±0.13）mg/g. 1.6 mg/mL extracts from Julypromoted adipocyte glucose uptake level to 1.66（±0.23）mmol/L，which was better than 8 U/mL insulin. Themaximum tolerated dose of guava leaves extracts in theacute toxicity test was more than 10 g/kg.