SIRT3, a NAD+-dependent protein deacetylase, regulates fatty acid oxidation by deacetylating mitochondrial proteins. During fasting, SIRT3 expression increases in liver and brown adipose tissues, leading to the deacetylation of long-chain acyl CoA dehydrogenase (LCAD). LCAD, a key enzyme in fatty acid oxidation, is hyperacetylated in SIRT3-deficient mice, reducing its enzymatic activity. This results in reduced fatty acid oxidation, increased triglyceride levels, and decreased ATP production in the liver. Mice lacking SIRT3 also exhibit cold intolerance and hypoglycemia during fasting, highlighting the importance of SIRT3 in maintaining metabolic homeostasis. The study identifies acetylation as a novel regulatory mechanism for mitochondrial fatty acid oxidation and suggests that SIRT3 modulates mitochondrial intermediary metabolism and fatty acid utilization during fasting.SIRT3, a NAD+-dependent protein deacetylase, regulates fatty acid oxidation by deacetylating mitochondrial proteins. During fasting, SIRT3 expression increases in liver and brown adipose tissues, leading to the deacetylation of long-chain acyl CoA dehydrogenase (LCAD). LCAD, a key enzyme in fatty acid oxidation, is hyperacetylated in SIRT3-deficient mice, reducing its enzymatic activity. This results in reduced fatty acid oxidation, increased triglyceride levels, and decreased ATP production in the liver. Mice lacking SIRT3 also exhibit cold intolerance and hypoglycemia during fasting, highlighting the importance of SIRT3 in maintaining metabolic homeostasis. The study identifies acetylation as a novel regulatory mechanism for mitochondrial fatty acid oxidation and suggests that SIRT3 modulates mitochondrial intermediary metabolism and fatty acid utilization during fasting.