Thyroid hormone receptors (TRs) play critical roles in energy homeostasis. To understand the role of TRs in lipid homeostasis in vivo, we adopted the loss-of-function approach by creating knock-in mutant mice with targeted mutation in the TRα gene (TRα1PV mouse) or TRβ gene (TRβPV mouse). The PV mutation, identified in a patient with resistance to thyroid hormone, exhibits potent dominant-negative activity. Here we show that in contrast to TRα1PV mouse, TRβPV mice exhibited no significant reduction in WAT but had significant increases in serum free fatty acids and total triglycerides. Moreover, the liver of TRβPV mice was markedly increased (33%) with excess lipid accumulation, but the liver mass of TRα1PV mouse was decreased (23%) with paucity of lipids. These results indicate that apo-TRβ and apo-TRα1 exerted distinct abnormalities in lipid metabolism. Further biochemical analyses indicate that increased lipogenic enzyme expression, activated peroxisome proliferator-activated receptor γ (Pparγ) signaling, and decreased fatty acid β-oxidation activity contributed to the adipogenic steatosis and lipid accumulation in the liver of TRβPV mice. In contrast, the expression of lipogenic enzymes and Pparγ was decreased in the liver of TRα1PV mice. These results suggest that the regulation of genes critical for lipid metabolism by TRs in the liver is isoform dependent. These results indicate that apo-TRβ and apo-TRα1 had different effects on lipid metabolism and that both TR isoforms contribute to the pathogenesis of lipid metabolism in hypothyroidism.