Hepatic ischemia-reperfusion (I/R) injury, a common clinical complication of liver transplantation, gravely affects patient prognosis. Krüppel-like factors (KLFs) constitute a family of C2/H2 zinc finger DNA-binding proteins. KLF6, a member of the KLF protein family, plays crucial roles in proliferation, metabolism, inflammation, and injury responses; however, its role in HIR is largely remains unknown. After I/R injury, we found that KLF6 expression in mice and hepatocytes was significantly upregulated. Mice were then subjected to I/R following injection of shKLF6- and KLF6-overexpressing adenovirus through the tail vein. KLF6 deficiency markedly exacerbated liver damage, cell apoptosis, and activation of hepatic inflammatory responses, whereas hepatic overexpression of KLF6 in mice produced the opposite results. In addition, we knocked out or overexpressed KLF6 in AML12 cells before exposing them to a hypoxia-reoxygenation challenge. KLF6 knockout decreased cell viability and increased hepatocyte inflammation, apoptosis, and ROS, whereas KLF6 overexpression had the opposite effects. Mechanistically, KLF6 inhibited the overactivation of autophagy at the initial stage, and the regulatory effect of KLF6 on I/R injury was autophagy-dependent. CHIP-qPCR and luciferase reporter gene assays confirmed that KLF6 bound to the promoter region of Beclin1 and inhibited its transcription. Additionally, KLF6 activated the mTOR/ULK1 pathway. Finally, we performed a retrospective analysis of the clinical data of liver transplantation patients and identified significant associations between KLF6 expression and liver function following liver transplantation. In conclusion, KLF6 inhibited the overactivation of autophagy via transcriptional regulation of Beclin1 and activation of the mTOR/ULK1 pathway, thereby protecting the liver from I/R injury. KLF6 is expected to serve as a biomarker for estimating the severity of I/R injury following liver transplantation.