<p>Fatty liver disease or steatosis affects millions of people, and research is required to develop effective treatments for the disease. In this study, we explored the functional activity of the miR-1914-5p in one-carbon metabolism (1CM) in our in vitro model of steatosis. In this cellular model, miR-1914-5p-inhibitor reduced energetic metabolite levels. Moreover, the motivation for this investigation was reinforced by bioinformatic analyses that revealed potential target sequences for miR-1914-5p on mRNAs of the 1CM. Co-cultures of hepatic cells (7 HepG2: 3 LX-2) transfected with miR-1914-5p mimics or inhibitor and then cultivated in high fatty media were used as our model for investigations. The results demonstrated that the miR inhibition reduced S-adenosylmethionine (SAMe) and increased adenosylhomocysteine (SAH) levels, which resulted in reduced global genomic DNA methylation and increased levels of histone H3/K4 methylation. Together, those mechanisms activated gene expression. Moreover, the miR-inhibitor-transfected cells increased the expression levels of relevant genes in the 1CM without activating RNA interference mechanisms. In addition, the miR-inhibitor controlled the levels of oxidative stress and the transsulfuration metabolism, potential deleterious metabolites produced by the 1CM metabolism. Opposing results were observed in cells transfected with the miR-1914-5 -mimics. Combined, the results suggests that the molecular changes observed in hepatic cells transfected with the miR-inhibitor indicated that this miR helps to control homeostasis and cellular survival in a steatotic environment, modulating the 1CM pathway, which is a nutrient sensor that integrates cellular metabolism, whose dysfunctions are frequently observed in fatty liver.</p>