The accumulation of intramyocellular lipid (IMCL) is recognized as an important determinant of insulin resistance, and is increased by a high-fat diet (HFD). However, the effects of HFD on IMCL and insulin sensitivity are highly variable. The aim of this study was to identify the genes in muscle that are related to this inter-individual variation. Fifty healthy men were recruited for this study. Before and after HFD for 3 days, IMCL levels in the tibialis anterior were measured by 1H-magnetic resonance spectroscopy, and peripheral insulin sensitivity was evaluated by glucose infusion rate (GIR) during the euglycemic-hyperinsulinemic clamp. Subjects who showed a large increase in IMCL and a large decrease in GIR by HFD were classified as high-responders (HRs), and subjects who showed a small increase in IMCL and a small decrease in GIR were classified as low-responders (LRs). In 5 subjects from each group, the gene expression profile of the vastus lateralis muscle was analyzed by DNA microarray analyses. Before HFD, gene expression profiles related to lipid metabolism were comparable between the 2 groups. Gene Set Enrichment Analysis demonstrated that 5 gene sets related to lipid metabolism were up-regulated by HFD in the HR group, but not in the LR group. Changes in gene expression patterns were confirmed by qRT-PCR using more samples (LR: n = 9; HR: n = 11). These results suggest that IMCL accumulation/impaired insulin sensitivity after HFD is closely associated with changes in the expression of genes related to lipid metabolism in muscle.