This study was conducted to evaluate the antihyperglycemic effect of an extract of Gynostemma pentaphyllum Makino, containing standardized concentrations of gypenosides, in C57BL/KSJ-db/db mice. For 5 weeks, animals were provided a standard AIN-76 diet (normal control) with rosiglitazone (0.005%, wt/wt) or two different doses of G. pentaphyllum ethanol extract (GPE) of the plant leaves (0.0025% and 0.01%, wt/wt). After the experimental period, the blood glucose levels of the high-dose GPE- and rosiglitazone-supplemented groups were significantly lower than that of the control group. The plasma insulin concentrations of the GPE-supplemented mice were significantly elevated compared to the control group. The GPE and rosiglitazone treatments profoundly affected the intraperitoneal insulin tolerance test compared to the control group, but not the intraperitoneal glucose tolerance test. In the evaluation of effects on hepatic glucose metabolism, the ratios of glucokinase/glucose-6-phosphatase activities in the high-dose GPE- and rosiglitazone-supplemented groups were prominently higher than that of the control group. The histology of the pancreatic islets revealed that the insulin-positive beta-cell numbers were higher in the high-dose GPE- and rosiglitazone-supplemented groups than in the control group. These results suggest that the supplementation of high-dose GPE (0.01%) in the diet lowers the blood glucose level by altering the hepatic glucose metabolic enzyme activities.