Increased hepatic glucose output is one of the major mechanisms of hyperglycemia in diabetic patients. Fructose-2,6-bisphosphate (F-2,6-BP), a gluconeogenic intermediate, plays a critical role in hepatic glucose output by regulating gluconeogenesis and glycolysis in the liver. Brazilin, an active component of sappan wood (Caesalpinia sappan), decreases blood glucose in diabetic animals. In this study, the effect of brazilin on gluconeogenic intermediate production and enzyme activity were examined to investigate the hypoglycemic mechanism of brazilin. Brazilin increased the production of F-2,6-BP in hepatocytes by elevating intracellular levels of fructose-6-phosphate (F-6-P) and hexose-6-phosphate (H-6-P). Brazilin was also found to significantly increase the activity of 6-phosphofructo-2-kinase (PFK-2) and pyruvate kinase in glucagon-treated hepatocytes. However, glucose-6-phosphatase activity was not affected by brazilin. This data suggests that brazilin inhibits hepatic gluconeogenesis by elevating the F-2,6-BP level in hepatocytes, possibly by elevating cellular F-6-P/H-6-P levels and PFK-2 activity. Increased pyruvate kinase activity may also play a role in the anti-gluconeogenic action of brazilin.