Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy‐related protein 7 (Atg7), Beclin‐1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin‐1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid–free and serum‐free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein–microtubule‐associated protein light chain 3 (GFP‐LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl‐Leu‐Leu‐Val‐Tyr‐7‐amino‐4‐methylcoumarin. A/R markedly decreased Atg7 and Beclin‐1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R‐induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP‐LC3 confirmed A/R‐induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusion: Calpain 2–mediated degradation of Atg7 and Beclin‐1 impairs mitochondrial autophagy, and this subsequently leads to MPT‐dependent hepatocyte death after A/R. (HEPATOLOGY 2008.)