ABSTRACTIt has been known that high alcohol-producingKlebsiella pneumoniae(HiAlcKpn) is one of causative agents of nonalcoholic fatty liver disease (NAFLD). However, how HiAlcKpnpromotes liver injury remains unclear. Recent findings suggest that DNA methylation might associate with the pathogenesis of NAFLD. Herein, the role of DNA methylation in HiAlcKpn-induced liver injury was investigated. Murine models of NAFLD were established in C57BL/6N wild-type mice by gavaging HiAlcKpnfor 8 weeks. The liver injury was assessed based on the liver histopathology and biochemical indicators. In addition, DNA methylation in hepatic tissue was assessed by using dot bolt of 5-mC. RNA sequencing analysis and whole-genome bisulfite sequencing (WGBS) analysis were also performed. HiAlcKpnsignificantly increased the activity of aspartate transaminase (AST), alanine transaminase (ALT), triglycerides (TGs), and glutathione (GSH), while hypomethylation was associated with liver injury in the experimental mice induced by HiAlcKpn. The GO and KEGG pathway enrichment analysis of the transcriptome revealed that HiAlcKpninduced fat metabolic disorders and DNA damage. The conjoint analysis of methylome and transcriptome showed that hypomethylation regulated related gene expression in signal pathways of lipid formation and circadian rhythm, includingRorαandArntl1genes, which may be the dominant cause of NAFLD induced by HiAlcKpn. Data suggest that DNA hypomethylation might play an important role in liver injury of NAFLD induced by HiAlcKpn. Which possibly provides a new sight for understanding the mechanisms of NAFLD and selecting the potential therapeutic targets.IMPORTANCEHigh alcohol-producingKlebsiella pneumoniae(HiAlcKpn) is one of causative agents of nonalcoholic fatty liver disease (NAFLD) and could induce liver damage. DNA methylation, as a common epigenetic form following contact with an etiologic agent and pathogenesis, can affect chromosome stability and transcription. We conjointly analyzed DNA methylation and transcriptome levels in the established murine models to explore the potential mechanisms for further understanding the role of DNA methylation in the liver damage of HiAlcKpn-induced NAFLD. The analysis of the DNA methylation landscape contributes to our understanding of the entire disease process, which might be crucial in developing treatment strategies.