Context/Objective: The aim of this study was to evaluate the regulation of the fuel partitioning and energy metabolism in skeletal muscle during lipid overfeeding in healthy men. Design/Participants/Intervention: Thirty-nine healthy volunteers were overfed for 56 days with a high-fat diet (3180 kJ/d). Energy metabolism (indirect calorimetry) was characterized in the fasting state and during a test meal before and at the end of the diet. Skeletal muscle biopsies were taken at day 0 and day 56. Main Outcome Measures: Change in gene expression, mitochondrial respiration, nicotinamide adenine dinucleotide (NAD(+)) content, and acetylation of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) in skeletal muscle was measured. Results: Overfeeding increased body weight (+2.6 kg) and fat mass concomitantly with a shift in the use of substrates as energy fuel toward preferential oxidation of carbohydrates instead of lipids. Changes in lipid metabolic gene expression supported this observation, with a reduction in pyruvate dehydrogenase kinase 4 expression that could be the consequences of decreased NAD(+) concentration and reduced deacetylase activity of the sirtuins, as supported by hyperacetylation of PGC-1 alpha after overfeeding. Interestingly, this reduction of the sirtuin PGC-1 alpha pathway was associated with increased mitochondrial gene expression and higher respiration rate under these conditions. Conclusion: Adaptation to lipid overfeeding and regulation of fuel partitioning in human muscle appear to rely on a dissociation between the regulatory functions of the sirtuin-PGC-1 alpha pathway on fatty acid oxidation and on mitochondrial regulation. This may facilitate lipid storage during a period of positive energy balance while maintaining mitochondrial functions and oxidative capacities.