Thyroid hormone has profound and diverse effects on liver metabolism. Here we show that tri-iodothyronine (T3) treatment in mice acutely and specifically induces hepatic expression of the metabolic regulator fibroblast growth factor 21 (FGF21). Mice treated with T3 showed a dose-dependent increase in hepatic FGF21 expression with significant induction at doses as low as 100 microg/kg. Time course studies determined that induction is seen as early as 4 h after treatment with a further increase in expression at 6 h after injection. As FGF21 expression is downstream of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha), we treated PPARalpha knock-out mice with T3 and found no increase in expression, indicating that hepatic regulation of FGF21 by T3 in liver is via a PPARalpha-dependent mechanism. In contrast, in white adipose tissue, FGF21 expression was suppressed by T3 treatment, with other T3 targets unaffected. In cell culture studies with an FGF21 reporter construct, we determined that three transcription factors are required for induction of FGF21 expression: thyroid hormone receptor beta (TRbeta), retinoid X receptor (RXR), and PPARalpha. These findings indicate a novel regulatory pathway whereby T3 positively regulates hepatic FGF21 expression, presenting a novel therapeutic target for diseases such as non-alcoholic fatty liver disease.