Fatty liver or steatosis refers to a histopathological condition in which an excess accumulation of lipids (primarily triglycerides) within hepatocytes occurs. The clinical signi?cance of fatty liver is generally thought to be one of the leading causes of hepatic dysfunction. Accumulating evidence indicates that impaired mitochondrial function plays a central role in the fatty liver. Previously we showed that 3,5-diiodothyronine (T2), a naturally occurring iodothyronine, when administered simultaneously with a high-fat diet (HFD), prevented excessive body weight gain and the development of liver steatosis by increasing hepatic fatty acid oxidation. These effects are correlated with a direct activation of sirtuin 1 (SIRT1) by T2. Here we studied the effect of T2 on mitochondrial SIRT3 target proteins activation in liver from HDF rats. We found that T2, contemporarily administered with the diet, caused deacetylation of the SIRT3 targets among which we identified long-chain-acylCoA dehydrogenase and 3-hydroxy-3methylglutaryl CoA synthase, associated with increased mitochondrial oxidative activity. Our results demonstrate that the beneficial effects of T2 supplementation on hepatic steatosis could be, at least in part, mediated by its ability to increase mitochondrial oxidative capacity through the deacetylation by SIRT3. Thus, sirtuins contribute to the T2-induced amelioration of obesity-related parameters such as hepatic steatosis.