In this study, effect of Osr1 deficiency in promoting non-alcoholic fatty liver disease progression was examined. The authors demonstrate that Osr1 regulates hepatic inflammation and cell survival through multiple signaling pathways and DNA methylation modification. Odd-skipped related 1 (Osr1) is a novel tumor suppressor gene in several cancer cell lines. Non-alcoholic steatohepatitis (NASH) is considered as a high-risk factor for hepatocellular carcinoma (HCC). This study is aimed to investigate the novel role of Osr1 in promoting the progression of hepatic steatosis to NASH. Following 12 weeks of diethylnitrosamine (DEN) and high-fat diet (HFD), wildtype (WT) and Osr1 heterozygous (Osr1(+/-)) male mice were examined for liver injuries.Osr1(+/-)mice displayed worsen liver injury with higher serum alanine aminotransferase levels than theWTmice. TheOsr1(+/-)mice also revealed early signs of collagen deposition with increased hepaticTgfbandFn1expression. There was overactivation of both JNK and NF-kappa B signaling in theOsr1(+/-)liver, along with accumulation of F4/80+ cells and enhanced hepatic expression ofIl-1bandIl-6. Moreover, theOsr1(+/-)liver displayed hyperphosphorylation of AKT/mTOR signaling, associated with overexpression ofBcl-2. In addition,Osr1(+/-)andWTmice displayed differences in the DNA methylome of the liver cells. Specifically, Osr1-responsible CpG islands ofCcl3andPcgf2, genes for inflammation and macrophage infiltration, were further identified. Taken together, Osr1 plays an important role in regulating cell inflammation and survival through multiple signaling pathways and DNA methylation modification for NAFLD progression.