Pioglitazone, a thiazolidinedone derivative, decreases insulin resistance and improves hyperglycemia in insulin-resistant obese and/or diabetic animals. However, the mechanisms by which hyperglycemia is improved are not well defined. We investigated the effects of pioglitazone on hepatic glucose metabolism using a perfused rat liver model. Perfusion with the buffer containing 1 – 10 µm pioglitazone for 20 min dose-dependently increased the hepatic fructose 2,6-bisphosphate content, a potent activator of 6-phosphofructo 1-kinase. The furctose 2,6-bisphosphate level after 20 min perfusion with 10 µm pioglitazone was 64.9± 14.5 pmol/mg ⋅ protein, significantly higher than the control (48.3±10.9 pmol/mg ⋅ protein). When the liver from a starved for 48 h rat was perfused with the buffer containing 2 mm lactate but no glucose, glucose was generated from lactate via the gluconeogenic pathway and flowed into the effluent perfusate at a constant rate of 31±0.6 µmol/g ⋅ liver/h. The addition of 10 µm pioglitazone decreased the glucose output rate to 19.3±3.8 µmol/g ⋅ liver/h. Dose-dependent inhibition of glucose output by pioglitazone was observed in the 1 – 10 µm dose range. These results indicate that pioglitazone may not only stimulate glycolysis but also inhibit gluconeogenesis in the liver. These acute and insulin-independent effects on hepatic glucose metabolism may partly account for the diverse anti-diabetic effects of pioglitazone.