Cholesterol-based but not plant sterol-based high-fat diet protects germfree (GF) mice from diet-induced obesity (DIO). DIO resistant GF mice show preferential carbohydrate oxidation, higher energy expenditure and energy and fat excretion. DIO resistance in GF mice is accompanied by increased steroid hormone levels but decreased bile acid levels in the cecum. Substrate oxidation and fat excretion in DIO resistant GF mice is linked to decreased hepatic Cyp7a1 and Nr1h4 expression.