Insulin regulates the rate of expression of many hepatic genes, including PEPCK, glucose-6-phosphatase (G6Pase), and glucose-6-phosphate dehydrogenase (G6PDHase). The expression of these genes is also abnormally regulated in type 2 diabetes. We demonstrate here that treatment of hepatoma cells with 5-aminoimidazole-4-carboxamide riboside (AICAR), an agent that activates AMP-activated protein kinase (AMPK), mimics the ability of insulin to repress PEPCK gene transcription. It also partially represses G6Pase gene transcription and yet has no effect on the expression of G6PDHase or the constitutively expressed genes cyclophilin or beta-actin. Several lines of evidence suggest that the insulin-mimetic effects of AICAR are mediated by activation of AMPK. Also, insulin does not activate AMPK in H4IIE cells, suggesting that this protein kinase does not link the insulin receptor to the PEPCK and G6Pase gene promoters. Instead, AMPK and insulin may lie on distinct pathways that converge at a point upstream of these 2 gene promoters. Investigation of the pathway by which AMPK acts may therefore give insight into the mechanism of action of insulin. Our results also suggest that activation of AMPK would inhibit hepatic gluconeogenesis in an insulin-independent manner and thus help to reverse the hyperglycemia associated with type 2 diabetes.