To define the role of mitochondrial glycerol-3-phosphate dehydrogenase (mGPD; EC 1.1.99.5) in energy balance and intermediary metabolism, we studied transgenic mice not expressing mGPD (mGPD−/−). These mice had ≈14% lower blood glucose; ≈50% higher serum glycerol; ≈80% higher serum triglycerides; and at thermoneutrality, their energy expenditure (Qo₂) was 15% lower than in wild-type (WT) mice. Glycerol-3-phosphate levels and lactate-to-pyruvate ratios were threefold elevated in muscle, but not in liver, of mGPD−/− mice. WT and mGPD−/− mice were then challenged with a high-fat diet, fasting, or food restriction. The high-fat diet caused more weight gain and adiposity in mGPD−/− than in WT female mice, without the genotype differentially affecting Qo₂ or energy intake. After a 30-h fast, WT female lost 60% more weight than mGPD−/− mice but these latter became more hypothermic. When energy intake was restricted to 50–70% of the ad libitum intake for 10 days, mGPD−/− female mice lost less weight than WT controls, but they had lower Qo₂ and body temperature. WT and mGPD−/− male mice did not differ significantly in their responses to these challenges. These results show that the lack of mGPD causes significant alterations of intermediary metabolism, which are more pronounced in muscle than liver and lead to a thrifty phenotype that is more marked in females than males. Lower T₄-to-T₃ conversion in mGPD−/− females and a greater reliance of normal females on mGPD to respond to high-fat diets make the lack of the enzyme more consequential in the female gender.