Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.