The aim of the study was to explore the effect of Gynostemma pentaphyllum (GP) extract on hepatic glucose output (HGO) in spontaneously type 2 diabetic Goto-Kakizaki (GK) rats treated orally with GP or placebo extract 1600 mg/kg daily, during three days or three weeks. The three-week treatment of GP, but not three-day treatment, significantly reduced plasma glucose (PG) levels from 9.8 ± 0.6 to 6.8 ± 0.4 mmol/L (p=0.027) in GK rats, whereas PG levels were not significantly decreased in the placebo rats. Glucose tolerance assessed by an intra-peritoneal glucose tolerance test was significantly improved in GP treated compared to placebo treated group (areas under the glucose curves, AUCs, from 0 to 120 min were 1150 ± 200 vs. 1761 ± 87 mmol/L; p=0.013). The glucose response in an intra-peritoneal pyruvate tolerance test from minute 15 to minute 120, the AUC (15-120) was significantly lower in the GP group (415.5 ± 68.0 vs. 641.5 ± 41.8 mmol/L; p<0.05). In liver perfusions, the AUCs for HGO during 18 min (0-18 min) were significantly decreased in GP treated rats compared with control rats (302.8 ± 36.5 vs. 423.5 ± 44.7 μmol, p<0.05). The three-week GP treatment significantly reduced the hepatic glycogen content, but not glycogen synthase activity compared to placebo group (p<0.007). In conclusion, three-week treatment of GP extract exerted anti-diabetic effect in GK rats, reducing plasma glucose levels and HGO, suggesting that GP improves the hepatic insulin sensitivity by suppressing gluconeogenesis.