Obesity is increasing in prevalence and has become a global public health problem. The main cause of obesity is a perturbation in energy homeostasis, whereby energy intake exceeds energy expenditure. Although mitochondrial dysfunction has been linked to the deregulation of energy homeostasis, the precise mechanism is poorly understood. Here, we identify mitochondrial p32 (also known as C1QBP) as an important regulator of lipid homeostasis that regulates both aerobic and anaerobic energy metabolism. We show that while whole-body deletion of the p32 results in an embryonic lethal phenotype, mice heterozygous for p32 are resistant to age- and high-fat diet-induced ailments, including obesity, hyperglycemia, and hepatosteatosis. Notably, p32 +/- mice are apparently healthy, demonstrate an increased lean-to-fat ratio, and show dramatically improved insulin sensitivity despite prolonged high-fat diet feeding. The p32 +/- mice show increased oxygen consumption and heat production, indicating that they expend more energy. Our analysis revealed that haploinsufficiency for p32 impairs glucose oxidation, which results in a compensatory increase in fatty acid oxidation and glycolysis. These metabolic alterations increase both aerobic and anaerobic energy expenditure. Collectively, our data show that p32 plays a critical role in energy homeostasis and represents a potential novel target for the development of anti-obesity drugs.