The low oxygen levels at high altitude are a potent and unavoidable physiological stressor to which highland mammals must adapt. One hypothesized adaptation to high altitude is an increased reliance on carbohydrates to support aerobic activities. Based on stoichiometries of combustion, ATP yield per mole of oxygen from carbohydrates is approximately 15% higher than from lipids [1, 2] (observed difference closer to 30% [3, 4]), and increased carbohydrate use represents an important oxygen-saving strategy that may be under high selective pressure. Although this hypothesis was first proposed nearly 30 years ago [5], the in vivo patterns of whole-body fuel use during exercise remain undefined for any highland mammal (including humans). Here we use a powerful multispecies approach to show that wild-caught high-altitude (4,000-4,500 m) native species of mice (Phyllotis andium and Phyllotis xanthopygus) from the Peruvian Andes use proportionately more carbohydrates and have higher oxidative capacities of cardiac muscles compared to closely related low-altitude (100-300 m) native counterparts (Phyllotis amicus and Phyllotis limatus). These results strongly infer that highland Phyllotis have evolved a metabolic strategy to economize oxygen when performing energy-demanding tasks at altitude. This study provides compelling evidence of adjustments in fuel use as an adaptation to high-altitude hypoxia in mammals. © 2012 Elsevier Ltd.