Our previous study found that thyroid-stimulating hormone promoted sterol regulatory element-binding protein-2 (SREBP-2) expression and suppressed AMP-activated protein kinase (AMPK) activity in the liver, but it was unclear whether there was a direct link between TSH, AMPK and SREBP-2. Here, we demonstrate that the 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced activation of AMPK directly inhibited the expression of SREBP-2 and its target genes HMGCR and HMGCS, which are key enzymes in cholesterol biosynthesis, and suppressed the TSH-stimulated up-regulation of SREBP-2 in HepG2 cells; similar results were obtained in TSH receptor knockout mice. Furthermore, AMPK, an evolutionally conserved serine/threonine kinase, phosphorylated threonine residues in the precursor and nuclear forms of SREBP-2, and TSH interacted with AMPK to influence SREBP-2 phosphorylation. These findings may represent a molecular mechanism by which AMPK ameliorates the hepatic steatosis and hypercholesterolemia associated with high TSH levels in patients with subclinical hypothyroidism (SCH).