Cholesterol homeostasis is strictly regulated through the modulation of HMG-CoA Reductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis. Phosphorylation of HMGCR inactivates and dephosphorylation activates it. AMPK is the major kinase phosphorylating the enzyme. Our previous study found that thyroid-stimulating hormone (TSH) increased the hepatocytic HMGCR expression, but it was still unclear if TSH affected hepatic HMGCR phosphorylation associated with AMPK. We used bTSH to treat the primary mouse hepatocytes and HepG2 cells with or without CA-AMPK plasmid or protein kinase A inhibitor (H89), and set up the TSH receptor (Tshr)-knockout mouse models. The p-HMGCR, p-AMPK and related molecular expression were tested. The ratios of p-HMGCR/HMGCR and p-AMPK/AMPK decreased in the hepatocytes in a dose-dependent manner following bTSH stimulation. The changes above were inversed when the cells were treated with CA-AMPK plasmid or H89. In Tshr-knockout mice, the ratios of liver p-HMGCR/HMGCR and p-AMPK/AMPK were increased relative to the littermate wild-type mice. Consistently, the phosphorylation of ACC, a downstream target molecular of AMPK, increased. All results suggested that TSH could regulate the phosphorylation of HMGCR via AMPK, which established a potential mechanism for hypercholesterolemia involved in a direct action of the TSH in liver.