OBJECTIVE: To investigate molecular adaptations that accompany the elevation of intramyocellular lipid (IMCL) content on a high-fat (HF) diet for 1 week. RESEARCH METHODS AND PROCEDURES: Ten subjects consumed a normal-fat (NF) diet for 1 week, followed by an HF diet for another week. After both dietary periods, we determined the IMCL content by proton magnetic resonance spectroscopy in the vastus lateralis muscle and quantified changes in gene expression, protein content, and activity in biopsy samples. We investigated genes involved in carbohydrate and fatty acid handling [lipoprotein lipase, acetyl-coenzyme A carboxylase (ACC) 2, hormone-sensitive lipase, hexokinase II, and glucose transporter 4] and measured protein levels of CD36 and phosphorylated and unphosphorylated ACC2 and the activity of adenosine monophosphate-activated kinase. RESULTS: IMCL content was increased by 54% after the HF period. Lipoprotein lipase mRNA concentration was increased by 33%, whereas ACC2 mRNA concentration tended to be increased after the HF diet. Hexokinase II, glucose transporter 4, and hormone-sensitive lipase mRNA were unchanged after the HF diet. ACC2 and CD36 protein levels, phosphorylation status of ACC2, and adenosine monophosphate-activated kinase activity did not change in response to the HF diet. DISCUSSION: We found that IMCL content in skeletal muscle increased after 1 week of HF feeding, accompanied by molecular adaptations that favor fat storage in muscle rather than oxidation.