SIRT3 regulates fatty acid oxidation via reversible enzyme deacetylation. SIRT3, a NAD+-dependent protein deacetylase localized in the mitochondrial matrix, regulates the acetylation levels of metabolic enzymes. Mice lacking both SIRT3 alleles appear normal under basal conditions but show marked hyperacetylation of mitochondrial proteins during fasting. SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. Mice lacking SIRT3 show higher levels of fatty acid oxidation intermediate products and triglycerides during fasting, associated with decreased fatty acid oxidation. Mass spectrometry analysis shows that long-chain acyl CoA dehydrogenase (LCAD) is hyperacetylated in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo, and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty acid utilization during fasting. Proteomics analysis of mitochondrial proteins revealed that the acetylation levels of numerous mitochondrial proteins change during fasting. The dependence of SIRT3 enzymatic activity on NAD+ suggests that SIRT3 could serve as a metabolic sensor and couples the energy status of the cell with the level of mitochondrial protein acetylation. SIRT3 expression was also upregulated in response to fasting in brown adipose tissue but not in the brain, heart or kidney. The liver is an important site of metabolic regulation under fasting conditions, and a metabolomic approach revealed multiple abnormalities in lipid metabolism products in SIRT3-/- mice. Long-chain acylcarnitine species accumulated in the liver, suggesting incomplete oxidation of long-chain fatty acids. Plasma acylcarnitine analysis revealed a striking positive relationship between the abundance of acylcarnitines and their chain length. SIRT3-/- mice showed increased urine methylsuccinate, ethylmalonate, and isobutyrylglycine, further supporting a defect in fatty acid oxidation. Biochemical tissue analysis revealed increased hepatic triglycerides in SIRT3-/- mice. Liver triglyceride levels were comparable under fed conditions between wt and SIRT3-/- mice. Triglyceride levels markedly increased during fasting in wt mice, consistent with the mobilization of fatty acids from adipose tissue to the liver. This accumulation was further exacerbated in mice lacking SIRT3, suggesting abnormal fatty acid metabolism. Hepatic steatosis is highly correlated with reduced lipid oxidation. To directly assess fatty acid oxidation, ex vivo palmitate oxidation was measured in liver homogenates from wt and SIRT3-/- mice. Under low substrateSIRT3 regulates fatty acid oxidation via reversible enzyme deacetylation. SIRT3, a NAD+-dependent protein deacetylase localized in the mitochondrial matrix, regulates the acetylation levels of metabolic enzymes. Mice lacking both SIRT3 alleles appear normal under basal conditions but show marked hyperacetylation of mitochondrial proteins during fasting. SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. Mice lacking SIRT3 show higher levels of fatty acid oxidation intermediate products and triglycerides during fasting, associated with decreased fatty acid oxidation. Mass spectrometry analysis shows that long-chain acyl CoA dehydrogenase (LCAD) is hyperacetylated in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo, and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty acid utilization during fasting. Proteomics analysis of mitochondrial proteins revealed that the acetylation levels of numerous mitochondrial proteins change during fasting. The dependence of SIRT3 enzymatic activity on NAD+ suggests that SIRT3 could serve as a metabolic sensor and couples the energy status of the cell with the level of mitochondrial protein acetylation. SIRT3 expression was also upregulated in response to fasting in brown adipose tissue but not in the brain, heart or kidney. The liver is an important site of metabolic regulation under fasting conditions, and a metabolomic approach revealed multiple abnormalities in lipid metabolism products in SIRT3-/- mice. Long-chain acylcarnitine species accumulated in the liver, suggesting incomplete oxidation of long-chain fatty acids. Plasma acylcarnitine analysis revealed a striking positive relationship between the abundance of acylcarnitines and their chain length. SIRT3-/- mice showed increased urine methylsuccinate, ethylmalonate, and isobutyrylglycine, further supporting a defect in fatty acid oxidation. Biochemical tissue analysis revealed increased hepatic triglycerides in SIRT3-/- mice. Liver triglyceride levels were comparable under fed conditions between wt and SIRT3-/- mice. Triglyceride levels markedly increased during fasting in wt mice, consistent with the mobilization of fatty acids from adipose tissue to the liver. This accumulation was further exacerbated in mice lacking SIRT3, suggesting abnormal fatty acid metabolism. Hepatic steatosis is highly correlated with reduced lipid oxidation. To directly assess fatty acid oxidation, ex vivo palmitate oxidation was measured in liver homogenates from wt and SIRT3-/- mice. Under low substrate