Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene () was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of was evaluated with the aid of an RT profiler-array. After shRNA-silencing of in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in -knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in -KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.