Better ways to prevent the cold ischemia-warm reperfusion (CI/WR) injury associated with liver transplantation are needed, and many investigations have focused on the molecular mechanisms of this injury. However, the mechanisms reported to date are controversial and no improvement in therapy has resulted. Here, using prolonged CI and orthotopic transplantation of rat liver grafts, we found that the CI/WR injury was closely associated with autophagy. By 15 minutes after the start of WR, small masses of hepatocytes that possessed abundant autophagosomes and autolysosomes frequently dissociated from the hepatic cords and obstructed the sinusoid, causing massive necrosis of hepatocytes within 2 hours. The cell masses included TUNEL-positive nuclei without caspase-3 and -7 activation. Autophagy suppression with the phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, reduced both liver damage and the mortality rate of recipient rats. To elucidate the downstream mechanisms of this autophagic pathway, liver grafts were treated with aspartic and cysteine proteinase inhibitors, pepstatin and leupeptin. This treatment also significantly improved the survival rate of recipient rats. These data suggest that autophagy-associated hepatocyte death triggers liver graft dysfunction. The protective effects of suppressing autophagy may suggest new ways to prevent CI/WR injury of the liver.