Abstract: The effects of 5-amino-4-imidazolecarboxamide riboside（AICAR）and the antidiabetic drug metformin on AMP-activated protein kinase (AMPK) activity and its downstream effector, acetyl-CoA carboxylase (ACC),β-hydroxy-β-methylglutary-CoA reductase（HMGR）activities in laying hens’ primary cultured hepatocytes were studied. Four treatments were employed in this experiment AICAR（0.5mmol/L）, metformin（0.5mmol/L）, and metformin（1 mmol/L）were added respectively into the standard culture medium(control). At 100 min after incubation, AICAR or metformin activated AMPK significantly ( P＜0.05), inactivated ACC and HMGR which are the major enzymes involved in fatty acid and cholesterol biosyntheses. AMPK regulated ACC and HMGR activities negatively. AICAR or metformin decreased hepatic lipid and cholesterol contents by activating AMPK. AMPK negatively regulated hepatic lipid and cholesterol contents. These findings suggest that the regulation of hepatic lipid and the cholesterol metabolism of primary hepatocytes in laying hens can be mediated by AMPK. 