Effects of endurance training on the phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme of gluconeogenesis, were studied in the obese Zucker rats. We used a moderate exercise program consisting of treadmill running at 20 m/min and 0-degree gradient for 1 h/day, 7 days/week, for 8 weeks. At the end of the experimental period, insulin action on glucose disposal rate was measured using the glucose-insulin index, the product of the areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test. Furthermore, changes of hepatic PEPCK gene expression were detected using reverse transcriptase polymerase chain reaction to assay the mRNA level and Western blot analysis to detect the protein level. Different to sedentary obese rats, an elevation in the value of glucose-insulin index from the exercised obese rats declined, indicating the marked effect of regular moderate exercise on the improvement of insulin sensitivity in this insulin resistant animal model. Moreover, the diabetes-related elevation in mRNA level and protein content of hepatic PEPCK were observed in non-exercise obese groups but they were markedly reduced by exercise training. In addition, chronic exercise training enhanced the insulin sensitivity of lean Zucker rats, since the value of glucose-insulin index was lower than that of untrained lean groups. Also, the hepatic PEPCK gene expressions both the mRNA and protein levels were reduced in exercised lean Zucker rats as compared with their sedentary littermates. These results suggest that modulation of hepatic PEPCK gene expression by chronic exercise training might be related to the enhancement of insulin sensitivity. Thus, endurance exercise training could aid in the prevention and/or treatment of individuals with insulin resistance.