The effects of high-fat (HF) feeding on gene expression in the small intestine were examined using obesity-resistant A/J mice and obesity-prone C57BL/6J (B6) mice. Both strains of mice were maintained on low-fat (LF, 5% fat) or high-fat (HF, 30% fat) diets for two weeks. Quantitative reverse transcription-PCR analysis revealed that lipid metabolism-related genes, including carnitine palmitoyltransferase (CPT)-1, liver fatty acid binding protein, pyruvate dehydrogenase kinase 4, and NADP⁺-dependent cytosolic malic enzyme were up-regulated by HF feeding in both strains of mice. The up-regulated gene expression levels were higher in A/J mice than in B6 mice, suggesting more active lipid metabolism in the small intestine of A/J mice. The prominent up-regulation of the lipid metabolism-related genes were specific to the small intestine; the expression levels were little or unchanged in the liver, muscle and white adipose tissue. The increase by HF feeding and predominant expression of the intestinal lipid metabolism-related genes in A/J mice were reflected in the enzyme activities; malic enzyme, CPT, and -oxidation activities were increased by HF feeding, and the up-regulated malic enzyme and CPT activities were significantly higher in obesity-resistant A/J mice in comparison with those in obesity-prone B6 mice. These findings suggest that intestinal lipid metabolism is associated with susceptibility to obesity.