Recent studies showed that germ-free (GF) mice are resistant to obesity when consuming a high-fat, high-carbohydrate Western diet. However, it remains unclear what mechanisms are involved in the antiobesity phenotype and whether GF mice develop insulin resistance and dyslipidemia with high-fat (HF) feeding. In the present study, we compared the metabolic consequences of HF feeding on GF and conventional (conv) C57BL/6J mice. GF mice consumed fewer calories, excreted more fecal lipids, and weighed significantly less than conv mice. GF/HF animals also showed enhanced insulin sensitivity with improved glucose tolerance, reduced fasting and nonfasting insulinemia, and increased phospho-Akt((Ser-473)) in adipose tissue. In association with enhanced insulin sensitivity, GF/HF mice had reduced plasma TNF-α and total serum amyloid A concentrations. Reduced hypercholesterolemia, a moderate accretion of hepatic cholesterol, and an increase in fecal cholesterol excretion suggest an altered cholesterol metabolism in GF/HF mice. Pronounced nucleus SREBP2 proteins and up-regulation of cholesterol biosynthesis genes indicate that enhanced cholesterol biosynthesis contributed to the cholesterol homeostasis in GF/HF mice. Our results demonstrate that fewer calorie consumption and increased lipid excretion contributed to the obesity-resistant phenotype of GF/HF mice and reveal that insulin sensitivity and cholesterol metabolism are metabolic targets influenced by the gut microbiota.