Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an energy conversion dynamic range of up to 100-fold, is an extreme case for evaluating the cellular balance of ATP production and consumption. This study examined the role of Ca²⁺ in the entire oxidative phosphorylation reaction network in isolated skeletal muscle mitochondria and attempted to extrapolate these results back to the muscle, in vivo. Kinetic analysis was conducted to evaluate the dose–response effect of Ca²⁺ on the maximal velocity of oxidative phosphorylation (V_maxO) and the ADP affinity. Force-flow analysis evaluated the interplay between energetic driving forces and flux to determine the conductance, or effective activity, of individual steps within oxidative phosphorylation. Measured driving forces [extramitochondrial phosphorylation potential (ΔG_ATP), membrane potential, and redox states of NADH and cytochromes b_H, b_L, c₁, c, and a,a₃] were compared with flux (oxygen consumption) at 37 °C; 840 nM Ca²⁺ generated an ∼2-fold increase in V_maxO with no change in ADP affinity (∼43 μM). Force-flow analysis revealed that Ca²⁺ activation of V_maxO was distributed throughout the oxidative phosphorylation reaction sequence. Specifically, Ca²⁺ increased the conductance of Complex IV (2.3-fold), Complexes I and III (2.2-fold), ATP production/transport (2.4-fold), and fuel transport/dehydrogenases (1.7-fold). These data support the notion that Ca²⁺ activates the entire muscle oxidative phosphorylation cascade, while extrapolation of these data to the exercising muscle predicts a significant role of Ca²⁺ in maintaining cellular energy homeostasis.