Mammalian hibernators, such as golden-mantled ground squirrels (Callospermophilus lateralis; GMGS), cease to feed while reducing metabolic rate and body temperature during winter months, surviving exclusively on endogenous fuels stored prior to hibernation. We hypothesized that mitochondria, the cellular sites of oxidative metabolism, undergo tissue-specific seasonal adjustments in carbohydrate and fatty acid utilization to facilitate or compliment this remarkable phenotype. To address this, we performed high-resolution respirometry of mitochondria isolated from GMGS liver, heart, skeletal muscle, and brown adipose tissue (BAT) sampled during summer (active), fall (prehibernation), and winter (hibernation) seasons using multi-substrate titration protocols. Mitochondrial phospholipid composition was examined as a postulated intrinsic modulator of respiratory function across tissues and seasons. Respirometry revealed seasonal variations in mitochondrial oxidative phosphorylation capacity, substrate utilization, and coupling efficiency that reflected the distinct functions and metabolic demands of the tissues they support. A consistent finding across tissues was a greater influence of fatty acids (palmitoylcarnitine) on respiratory parameters during the prehibernation and hibernation seasons. In particular, fatty acids had a greater suppressive effect on pyruvate-supported oxidative phosphorylation in heart, muscle and liver mitochondria, and enhanced uncoupled respiration in BAT and muscle mitochondria in the colder seasons. Seasonal variations in the mitochondrial membrane composition reflected changes in the supply and utilization of polyunsaturated fatty acids, but were generally mild and inconsistent with functional variations. In conclusion, mitochondria respond to seasonal variations in physical activity, temperature and nutrient availability in a tissue-specific manner that compliment circannual shifts in the bioenergetic and thermoregulatory demands of mammalian hibernators.