Cholestenoic acid (CA) has been reported as an important biomarker of many severe diseases, but its physiological and pathological roles remain unclear. The study aimed to investigate the potential role of CA in hepatic lipid homeostasis. Enzyme kinetic studies revealed that CA specifically activates DNA methyltransferases 1 (DNMT1) at low concentration with EC50 = 1.99 × 10-6 M and inhibits the activity at higher concentration with IC50 = 9.13 × 10−6 M, and specifically inhibits DNMT3a, and DNMT3b activities with IC50= 8.41 × 10−6 M and IC50= 4.89 × 10−6 M, respectively. In a human hepatocyte in vitro model of high glucose-induced lipid accumulation, CA significantly increased demethylation of 5mCpG in the promoter regions of over 7,000 genes, particularly those involved in master signaling pathways such as calcium-AMPK and NAFLD. RNA sequencing analysis showed that the downregulated genes affected by CA encoding key enzymes, such as PCSK9, MVK, and HMGCR, which are involved in cholesterol metabolism and steroid biosynthesis pathways. Additionally, untargeted lipidomic analysis showed that CA significantly reduced neutral lipid levels by 60% in the cells cultured in high glucose media. Administration of CA in mouse MASLD models significantly decreases lipid accumulation, suppresses the gene expression involved in lipid biosynthesis in liver tissues, and alleviates liver function. This study shows that CA as an endogenous epigenetic regulator decreases lipid accumulation via epigenetic regulation. The results indicate that CA can be considered a potential therapeutic target for the treatment of metabolic disorders.