Fasting induces profound changes in the hypothalamus-pituitary-thyroid axis and peripheral thyroid hormone (TH) metabolism, ultimately leading to lower serum thyroid hormone (TH) concentrations. In the present study, we aimed to investigate the regulation of type 3 deiodinase (D3) during fasting in two metabolic tissues: liver and white adipose tissue (WAT). To this end, we studied the effect of modulation of the mammalian target of rapamycin (mTOR) and hypoxia inducible factor 1α (HIF1α) on D3 expression in a primary rat hepatocytes and in 3T3-L1 adipocytes. In addition, we studied the role of the constitutive androstane receptor (CAR) on liver TH metabolism using primary hepatocytes and CAR-/- mice. Twenty-four hour fasting increased liver D3 expression in mice. Inhibition of mTOR using mTOR inhibitors markedly induced D3 mRNA expression in primary hepatocytes; this increase was accompanied by a small increase in D3 activity. Stimulation of these cells with a CAR agonist induced both D3 mRNA expression and activity. Fasting increased hepatic D3 expression in WT but not in CAR-/- mice. In WAT, D3 mRNA expression increased 5-fold after 48h fasting. Treatment of 3T3-L1 adipocytes with mTOR inhibitors induced D3 mRNA expression, whereas stimulation of these cells with cobalt chloride, a compound that mimics hypoxia and stabilizes HIF1α, did not induce D3 mRNA expression. In conclusion, our results indicate an important role of mTOR in the upregulation of D3 in WAT and liver during fasting. Furthermore, CAR plays a role in the fasting induced D3 increase in the liver.