Volatile anesthetics protect against cardiac ischemia/perfusion (IR) injury. Excess mitochondrial free Ca2+ ([Ca2+]m) is a major cause of mitochondria ‐mediated IR injury. To better understand mechanisms of anesthetic ‐mediated protection of mitochondrial function, we monitored time courses of NADH redox state, respiration, ΔΨm, and matrix Ca2+ uptake/efflux kinetics during anesthetic exposure. Isolated mitochondria from rat hearts were energized with 10 mM Na+‐or K+‐pyruvate+malate, or with Na+‐succinate followed by isoflurane (0.5 to 2 mM), CaCl2 (≈ 200 nM free Ca2+), and ADP (250 μM). Our data showed that isoflurane: (a) increased [Ca2+]m despite ΔΨm depolarization, while decreasing Na+‐dependent Ca2+ efflux (NCE), (b) decreased state 3 NADH oxidation and increased state 3 duration with Na+‐pyruvate/malate, but c) caused no change in state 3 NADH oxidation but reduced state 3 duration with Na+‐succinate. These data indicate that isoflurane (1) increases [Ca2+]m by attenuating activity of NCE and, (2) decreases the rate of ADP phosphorylation while reducing the duration of state 3 respiration by attenuating complex I activity. Isoflurane's effects on inhibiting NCE and complex I together may act as the protective mechanism against IR injury at the mitochondrial level.