OBJECTIVES: Ischemia/reperfusion is a leading cause of liver damage after surgical intervention, trauma, and transplantation. It has been reported that the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 attenuates myocardial, cerebral, and renal ischemia/reperfusion damage. This study aimed to investigate the involvement of sirtuin 1-mediated p66shc inhibition in liver ischemia/reperfusion and explore the effect of carnosic acid and ischemic preconditioning on liver ischemia/reperfusion-induced damage. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats and HepG2 cells. INTERVENTIONS: The rats were subjected to 45 minutes of ischemia to 70% of the liver, followed by 3-hour reperfusion. The HepG2 cells were subjected to hypoxia/reoxygenation-induced injury. MEASUREMENTS AND MAIN RESULTS: In the rats with liver ischemia/reperfusion injury, carnosic acid pretreatment and ischemic preconditioning dramatically reduced the serum aminotransferase activity and proinflammatory chemokine levels and improved the liver histological evaluations. Carnosic acid and ischemic preconditioning also increased manganese superoxide dismutase and Bcl-xL, but down-regulated cleaved caspase-3. Interestingly, the protective effect of carnosic acid and ischemic preconditioning was positively associated with sirtuin 1 activation. By contrast, p66shc, a kinase that promotes oxidative injury and apoptosis, was inhibited by carnosic acid and ischemic preconditioning. Sirtuin 1 small interfering RNA knockdown experiments confirmed that carnosic acid increased sirtuin 1-mediated repression of p66shc in HepG2 cells and that the protective effect of carnosic acid against hypoxia/reoxygenation injury was inhibited by the sirtuin 1 inhibitor nicotinamide. These results suggest that carnosic acid protects hepatocytes from hypoxia/reoxygenation damage through sirtuin 1-mediated p66shc suppression. To support this notion, we further demonstrated that the sirtuin 1 activator resveratrol achieved a protective effect similar to that of carnosic acid against hypoxia/reoxygenation injury, whereas sirtuin 1 small interfering RNA and nicotinamide had the opposite effect. CONCLUSIONS: Carnosic acid and ischemic preconditioning protect against ischemia/reperfusion-induced liver injury. Mechanistically, the protective effect involves the sirtuin 1-mediated inhibition of p66shc, suggesting that this pathway is a novel potential therapeutic target for protecting the liver from ischemia/reperfusion injury.