We report the production and metabolic phenotype of a knockout mouse line in which the Fmo5 gene has been disrupted. In comparison with wild-type (WT) mice, Fmo5-/- mice exhibit a lean phenotype, which is age-related, becoming apparent after 20 weeks of age. Despite greater food intake, Fmo5-/- mice weigh less, store less fat in white adipose tissue (WAT) and have lower plasma glucose and cholesterol concentrations. Fmo5-/- mice have enhanced whole-body energy expenditure, due mostly to increased resting energy expenditure, with no increase in physical activity. An increase in the respiratory exchange ratio (RER) during the dark phase, the period in which the mice are active, indicates a switch from fat to carbohydrate oxidation. In comparison with WT mice, the rate of fatty acid oxidation in Fmo5-/- mice is higher in WAT, which would contribute to depletion of lipid stores in this tissue, and lower in skeletal muscle, which would contribute to the higher RER of these animals. Five proteins were down regulated in the liver of Fmo5-/- mice: aldolase B, glycerol 3-phosphate dehydrogenase and ketohexokinase are involved in glucose or fructose metabolism; HMG-CoA synthase 1 is involved in cholesterol biosynthesis; and malic enzyme 1 catalyzes the oxidative decarboxylation of malate to pyruvate, in the process producing NADPH for use in lipid and cholesterol biosynthesis, thus providing a potential explanation for the reduced fat deposits and lower plasma cholesterol characteristic of Fmo5-/- mice. Our results indicate that disruption of the Fmo5 gene slows metabolic ageing via pleiotropic effects.