Ischemia-reperfusion (I/R) injury is a commonly encountered clinical problem in liver surgery and transplantation. The pathogenesis of I/R injury is multifactorial, but mitochondrial Ca²⁺ overload plays a central role. We have previously defined a novel pathway for mitochondrial Ca²⁺ handling and now further characterize this pathway and investigate a novel Ca²⁺-channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB), for preventing hepatic I/R injury. The effect of 2-APB on cellular and mitochondrial Ca²⁺ uptake was evaluated in vitro by using ⁴⁵Ca²⁺. Subsequently, 2-APB (2 mg/kg) or vehicle was injected into the portal vein of anesthetized rats either before or following 1 h of inflow occlusion to 70% of the liver. After 3 h of reperfusion, liver injury was assessed enzymatically and histologically. Hep G2 cells transfected with green fluorescent protein-tagged cytochrome c were used to evaluate mitochondrial permeability. 2-APB dose-dependently blocked Ca²⁺ uptake in isolated liver mitochondria and reduced cellular Ca²⁺ accumulation in Hep G2 cells. In vivo I/R increased liver enzymes 10-fold, and 2-APB prevented this when administered pre- or postischemia. 2-APB significantly reduced cellular damage determined by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of liver tissue. In vitro I/R caused a dissociation between cytochrome c and mitochondria in Hep G2 cells that was prevented by administration of 2-APB. These data further establish the role of cellular Ca²⁺ uptake and subsequent mitochondrial Ca²⁺ overload in I/R injury and identify 2-APB as a novel pharmacological inhibitor of liver I/R injury even when administered following a prolonged ischemic insult.