Injury of liver by ischaemia crucially involves mitochondrial damage. The role of Ca²⁺ in mitochondrial damage is still unclear. We investigated the effect of low micromolar Ca²⁺ concentrations on respiration, membrane permeability, and antioxidative defence in liver mitochondria exposed to hypoxia/reoxygenation. Hypoxia/reoxygenation caused decrease in state 3 respiration and in the respiratory control ratio. Liver mitochondria were almost completely protected at about 2 µm Ca²⁺. Below and above 2 µm Ca²⁺, mitochondrial function was deteriorated, as indicated by the decrease in respiratory control ratio. Above 2 µm Ca²⁺, the mitochondrial membrane was permeabilized, as demonstrated by the sensitivity of state 3 respiration to NADH. Below 2 µm Ca²⁺, the nitric oxide synthase inhibitor nitro‐l‐arginine methylester had a protective effect. The activities of the manganese superoxide dismutase and glutathione peroxidase after hypoxia showed maximal values at about 2 µm Ca²⁺. We conclude that Ca²⁺ exerts a protective effect on mitochondria within a narrow concentration window, by increasing the antioxidative defence.