Abstract: This study focused on the molecular mechanism of inhibition of activities of α-glucosidase and HMG-CoA reductase which were the key enzymes of hyperglycemia and hyperlipidemia by steviol and isosteviol. The inhibition rate of steviol and isosteviol to α-glucosidaseand HMG-CoA reductase was determined by spectrophotometry. The enzyme inhibition kinetics were studied by double reciprocal plot method, and the molecular binding mode was analysised by AutoDock software. The results showed that the IC₅₀ of steviol, isosteviol to α-glucosidase were 70.75 and 49.65 mg/L respectively. The activity of α-glucosidase was inhibited by competitive and non competitive methods. The IC₅₀ of steviol, isosteviol to HMG-CoA reductase were 46.29 and 36.66 mg/L, which competitively inhibited HMG-CoA reductase. The results of molecular docking showed that steviol and isosteviol were located in the hydrophobic pocket of α-glucosidase and HMG CoA reductase, they bind with multiple amino acid residues and have hydrophobic effect. This study would provide theoretical reference for the development of new type inhibitors of foodborne α-glucosidase and HMG-CoA reductase, and the promotion of the application of steviol and isosteviol in food and medicine.