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Objective: To investigate the effect and mechanism of antihyperglycemic agent metformin on the gene expression of phosphoenolpyruvate carboxykinase (PEPCK)-a key enzyme within the regulation of gluconeogenesis in hepatocytes and to determine whether the effects of metformin on hepatocytes are transmitted throughout the known insulin signaling pathways., Methods: Confluent H4IIE rat heptoma cells were cultured with metformin 0.1 mmol/L for 16 h and then stimulated with various agents: insulin, cyclic adenosine monophosphate (cAMP), dexamethasone, signaling transduction inhibitor wortmannin and UO126. The gene expression of PEPCK was examined by Northern blot analysis., Results: Metformin significantly decreased basal PEPCK mRNA levels by 75% (P < 0.01). Incubation with metformin alone had no significant effect on cAMP/dexamethasone stimulated PEPCK gene expression. In contrast, insulin 0.1 nmol/L significantly inhibited cAMP/dexamethasone stimulated PEPCK gene expression by 67% (P < 0.01), when insulin 0.1 nmol/L was present together with metformin, cAMP/dexamethasone induced PEPCK gene expression was decreased by 94% (P < 0.01). Both insulin signaling pathway inhibitors, phosphatidyl-inositol-3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase (MAPK) inhibitor UO126, had no significant effect on inhibited PEPCK mRNA expression by metformin, but wortmannin blocked significantly inhibitory regulation of insulin on PEPCK gene expression (P < 0.01)., Conclusion: Metformin can inhibit the PEPCK gene expression via either an insulin-independent or interaction with insulin manner. The inhibitory effects of insulin on PEPCK gene expression are mediated through PI3K pathway, not MAPK pathway.