Folic Acid Supplementation Attenuates Hepatic Steatosis by Enhancing Choline Availability and Remodeling Fatty Acid Profiles in Mice Fed a High‐Fat Diet

High-fat diets (HFDs) are a well-established cause of hepatic steatosis, a condition associated with altered hepatic fatty acid metabolism and reduced choline availability. Folic acid (FA) deficiency can also promote steatosis, in part by impairing choline metabolism. Although FA supplementation has been found to reduce liver fat in mice with hepatic steatosis, it is unclear if this effect is due to increased hepatic choline levels, changes in fatty acid profiles, or a combination of both. In this study, four-week-old male C57BL/6J mice were fed 45 kcal% HFDs with total FA content adjusted to onefold, fivefold, or tenfold AIN-93G recommended level (2 mg/kg diet) for 15 weeks ad libitum. Hepatic triacylglycerol (TAG), choline concentrations, expression of key genes in choline metabolism, and TAG-bound fatty acid profiles were analyzed. Mice receiving tenfold FA had lower liver weight and hepatic TAG levels compared to the onefold control group (<i>p</i> < 0.05). Both fivefold and tenfold FA supplementation increased hepatic choline concentrations and upregulated mRNA expression of choline-metabolizing genes (<i>p</i> < 0.05), suggesting enhanced choline utilization. Additionally, tenfold FA supplementation altered the hepatic TAG fatty acid profile, reducing levels of palmitoleic acid and oleic acid (<i>p</i> < 0.05), fatty acids typically associated with <i>de novo</i> lipogenesis. A strong inverse correlation was observed between hepatic choline and TAG levels (<i>p</i> < 0.001, adjusted <i>R</i> ² = 0.56), supporting a potential role for choline availability in mediating FA's protective effects. Folic acid supplementation protects against hepatic steatosis by enhancing choline availability, modulating lipid metabolism, and reducing liver fat accumulation.