Therapeutic hypothermia is a strategy that reduces metabolic rate and brain damage during clinically-relevant hypoxic events. Mitochondrial respiration is compromised by hypoxia, with deleterious consequences for the mammalian brain; however, little is known about the effects of reduced temperature on mitochondrial metabolism. Therefore, we examined how mitochondrial function is impacted by temperature using high resolution respirometry to assess electron transport system (ETS) function in saponin-permeabilized mouse brain at 28 and 37°C. Respirometric analysis revealed that, at the colder temperature, ETS respiratory flux was ~ 40-75% lower relative to the physiological temperature in all respiratory states and for all fuel substrates tested. In whole brain tissue, the enzyme maximum respiratory rates for complexes I-V were similarly reduced by between 37-88%. Complexes II and V were particularly temperature-sensitive; a temperature-mediated decrease in complex II activity may support a switch to complex I mediated ATP-production, which is considerably more oxygen-efficient. Finally, the mitochondrial H+-gradient was more tightly coupled, indicating that mitochondrial respiration is more efficient at the colder temperature. Taken together, our results suggest that improvements in mitochondrial function with colder temperatures may contribute to energy conservation and enhance cellular viability in hypoxic brain.