In skeletal muscle, resting intracellular Ca²⁺ concentration ([Ca²⁺]i) homeostasis is exquisitely regulated by Ca²⁺ transport across the sarcolemmal, mitochondrial and sarcoplasmic reticulum (SR) membranes. Of these three systems, the relative importance of the mitochondria in [Ca²⁺]i regulation remains poorly understood in in vivo skeletal muscle. We tested the hypothesis that the capacity for Ca²⁺ uptake by mitochondria is a primary factor in determining [Ca²⁺]i regulation in muscle at rest and following contractions. Tibialis anterior muscle of anesthetized PGC-1α overexpressing (PGC-1α OE, increased mitochondria model) and wild-type littermates (WT) mice were exteriorized in vivo and loaded with Fura-2 AM and Rhod-2 AM. Ca²⁺ buffering and mitochondrial [Ca²⁺] were evaluated at rest and during recovery from fatiguing tetanic contractions. In addition, the effects of pharmacological inhibition of SR (thapsigargin) and mitochondrial (FCCP) function were examined at rest. [Ca²⁺]i in WT remained elevated for the entire post-contraction recovery period but in PGC-1α OE [Ca²⁺]i returned to resting baseline. Thapsigargin immediately and substantially increased resting [Ca²⁺]i in WT whereas in PGC-1α OE this effect was delayed and markedly diminished. FCCP abolished this improvement of [Ca²⁺]i regulation in PGC-1α OE. Mitochondrial [Ca²⁺] accumulation was observed in PGC-1α OE following contractions and thapsigargin treatment. In the SR PGC-1α OE down-regulated SERCA1 (Ca²⁺ uptake) and PV (Ca²⁺ buffering) protein levels whilst mitochondrial Ca²⁺ uptake-related proteins (Mfn1, Mfn2 and MCU) were up-regulated. These data demonstrate a heretofore unappreciated role for skeletal muscle mitochondria in [Ca²⁺]i regulation in vivo following fatiguing tetanic contractions and at rest.