Hepatic preconditioning has emerged as a promising strategy of activating natural pathways to augment tolerance to liver ischemia-reperfusion (IR) injury. Liver-resident natural killer T (NKT) cells play an important role in modulating the local immune and inflammatory responses. This work was aimed to investigate whether preactivation of NKT cells could provide a beneficial “preconditioning” effect to ameliorate the subsequent hepatic IR injury. To selectively activate NKT cells, C57BL/6 mice were treated intraperitoneally with the glycolipid antigen α-galactosylceramide (α-GalCer) 1 h prior to hepatic ischemia. Significantly reduced liver IR injury was observed in mice pretreated with α- GalCer, and this protective effect was specifically abrogated by a CD1d blocking antibody. Serum TNF-α, IFN-γ, and IL-13 levels were markedly increased shortly after α-GalCer injection. Pretreatment with a neutralizing antibody against TNF-α or IFN-γ did not influence the protective effect of α-GalCer preconditioning, whereas preadministration of an IL-13 neutralizing antibody completely abolished the effect. Treatment with α-GalCer also led to an increased expression of adenosine A_2A receptor (A_2AR) in the liver, and blockade of A_2AR by SH58261 diminished α-GalCer pretreatment-mediated attenuation of liver IR injury. In contrast, administration of the selective A_2AR agonist CGS21680 reversed the counteracting effect of the IL-13 neutralizing antibody on α-GalCer preconditioning. Additionally, α-GalCer pretreatment was associated with a decreased neutrophil accumulation in the ischemic liver. These findings provide the first evidence that hepatic preconditioning by preactivation of NKT cells with α-GalCer protects the liver from IR injury via an IL-13 and adenosine A_2AR-dependent mechanism.