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Isoflurane has a pharmacological preconditioning effect against ischemia injury in the heart, kidney, and brain, but whether and how isoflurane preconditioning protects livers against ischemia and reperfusion (IR) injury is unclear.Mice were randomly divided into an isoflurane preconditioning (ISO) group and control group, receiving 1.5% isoflurane or carrier gas for 40 minutes, respectively (n = 8/group). A partial warm liver IR model was used, and liver injury was evaluated. Primary hepatocytes were pretreated with 1.5% isoflurane for 2 hours before the induction of cell death by hydrogen peroxide. Cell death and survival were evaluated with the lactate dehydrogenase and cell counting kit-8 assay. Autophagy and regulatory molecules in stressed livers and hepatocytes were analyzed by Western blot (n = 6/group). An autophagy inhibitor (3-methyladenine [3-MA]) and 5′ adenosine monophosphate-activated protein kinase (AMPK) inhibitor (dorsomorphin) were administered in vivo (n = 8/group) and in vitro (n = 6/group).Compared to that observed in the control group, mice in the ISO group showed reduced liver injury (alanine aminotransferase [ALT] levels, control versus ISO group, 8285 ± 769 vs 4896 ± 917 U/L, P < .001) and enhanced hepatocellular antiapoptosis in livers after IR. Furthermore, liver autophagy was restored by ISO as indicated by elevated LC3B II protein levels accompanied with increased p62 degradation. The in vitro study of primary hepatocytes also found that ISO effectively attenuated hepatocyte cell death induced by hydrogen peroxide. In addition, 3-MA pretreatment showed no significant influence in the control group, but abrogated the protective role of ISO both in stressed livers (ALT levels, phosphate-buffered saline + ISO versus 3-MA + ISO group, 5081 ± 294 vs 8663 ± 607 U/L, P < .001) and in hepatocytes. Finally, signaling pathway analysis demonstrated that AMPK was activated by ISO. Pretreatment with an AMPK inhibitor also abrogated liver protection by ISO (ALT levels, phosphate-buffered saline + ISO versus dorsomorphin [DOR] + ISO group, 5081 ± 294 vs 8710 ± 500 U/L, P < .001), with no significant effect in control mice.Our results indicate that isoflurane preconditioning attenuates liver IR injury via AMPK/mTOR-mediated hepatocellular autophagy restoration. Our findings provide a novel potential therapeutic strategy for managing liver IR injury.