Introduction: Cordycepin has been reported to exhibit hepatic protective and anti-inflammatory properties. Here, we investigated the role of cordycepin in ischemia/reperfusion (IR)-induced liver injury in a mouse model.Methods: Mice were pretreated with cordycepin by gavage for 3 weeks, followed by the establishment of the IR modeling. Liver injury, Suzuki's histological grading, hepatic apoptosis, and inflammatory responses were evaluated by biochemical and pathological analysis.Results: It was found that Cordycepin pretreatment at 50 mg/kg for 3 weeks attenuated IR-induced liver injury, as reflected by the significant decrease of the levels of aspartate aminotransferase, alanine transaminase, lactate dehydrogenase, and low-density lipoprotein. Cordycepin pretreatment also reduced histopathological changes, attenuated hepatocyte apoptosis, inflammatory responses in the livers of IR mice. Mechanically, toll-like receptor 4/nuclear factor kappa-B signaling in liver tissues was inhibited by Cordycepin pretreatment.Conclusions: In conclusion, Cordycepin pretreatment protects IR-induced liver injury, which demonstrates its potential for the treatment of IR in the liver.