Adiponectin plays important roles in regulating insulin sensitivity and atherogenesis. Adiponectin has been shown to suppress hepatic glucose production in rodents. It has not been reported whether ectopically expressed adiponectin could regulate glucose metabolism in cultured hepatocyte-like cells. In the current study, the effect of adiponectin on glucose production was analyzed by ectopically expressing the protein in hepatoma H4IIE cells using an adenovirus delivery system to generate both human full-length and the globular domain of the protein. Expression of adiponectin in hepatoma H4IIE cells, in the absence of insulin, suppressed expression of the genes encoding glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, rate-limiting enzymes in the gluconeogenic pathway. Furthermore, expression of adiponectin in H4IIE cells suppressed glucose production from lactate and pyruvate. Purified recombinant human adiponectin also reduced glucose production in H4IIE cells and in rat primary hepatocytes in the absence of insulin. These data suggest that adiponectin protein could exert its function independent of the presence of insulin in these culture systems.