The substantial rise in the prevalence of nonalcoholic steatohepatitis (NASH), an advanced form of nonalcoholic fatty liver disease, and the strong association between NASH and the development of hepatocellular carcinoma indicate the urgent need for a better understanding of the underlying mechanisms. In the present study, by using the Stelic animal model of NASH and NASH-derived liver carcinogenesis, we investigated the role of the folate-dependent 1-carbon metabolism in the pathogenesis of NASH. We demonstrated that advanced NASH and NASH-related liver carcinogenesis are characterized by a significant dysregulation of 1-carbon homeostasis, with diminished expression of key 1-carbon metabolism genes, especially a marked inhibition of the S-adenosylhomocysteine hydrolase (Ahcy) gene and an increased level of S-adenosyl-l-homocysteine (SAH). The reduction in Ahcy expression was associated with gene-specific cytosine DNA hypermethylation and enrichment of the gene promoter by trimethylated histone H3 lysine 27 and deacetylated histone H4 lysine 16, 2 main transcription-inhibiting markers. These results indicate that epigenetically mediated inhibition of Ahcy expression may be a driving force in causing SAH elevation and subsequent downstream disturbances in transsulfuration and transmethylation pathways during the development and progression of NASH.—Pogribny, IP, Dreval, K., Kindrat, I., Melnyk, S., Jimenez, L., de Conti, A., Tryndyak, V., Pogribna, M., Ortega, JF, James, SJ, Rusyn, I., Beland, FA Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.