It is known that high-fat diets (HFD) induce insulin resistance. Previously we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented body-weight and adipose-mass gain. The action of T2 was associated with the activity of AMP-activated protein kinase (AMPK). In this study we found that T2 prevented hepatic fat accumulation and stimulated mitochondrial fatty acid oxidation within 6 hours, increasing nuclear SIRT activity. When animals were concomitantly treated overnight with T2 and the SIRT1 inhibitor EX-527, mitochondrial fatty acid oxidation was inhibited. Furthermore, T2 treatment caused deacetylation of two key SIRT1 targets: peroxisome proliferator activated receptor (PPAR)? coactivator (PGC)-1alpha (involved in mitochondrial biogenesis and activity), and sterol regulatory element-binding protein (SREBP)-1c (involved in lipogenesis). Accordingly, after 2 weeks T2 upregulated PGC-1alpha target genes, and downregulated SREBP-1c target genes. T2 increased glucose tolerance and insulin sensitivity, reflected by downregulation of gluconeogenic genes. Only after 4 weeks of treatment phosphorylation of AMPK increased in the HFD-T2 animals, without changes in gene expression. T2, not other tested thyroid hormone metabolites, directly activated purified SIRT1 in vitro. Thus, T2, by activating SIRT1, prevents fat accumulation and diet-induced insulin resistance.