Using a high-fat diet-induced obesity-prone(OP) and obesity-resistant(OR) mouse model, combined with an integrated analysis of the liver transcriptome and fecal metabolome, we screened for differentially expressed genes(DEGs) and differential metabolites. Enzyme-linked immunosorbent assays(ELISA) were employed to detect differences in folic acid content in the serum and feces of mice. Furthermore, the role of folic acid in the adipogenic differentiation of 3T3-L1 preadipocytes was preliminarily investigated. Folic acid intervention in 3T3-L1 preadipocytes, gene expression assays, and molecular docking simulations were subsequently conducted to elucidate the regulatory function of folic acid on preadipocyte adipogenic differentiation. The results demonstrated that DEGs in the livers of OR mice were significantly enriched in the folic acid biosynthesis pathway, and folic acid was identified as a differential fecal metabolite, suggesting that folic acid participates in the progression of obesity by regulating lipid metabolism. Folic acid levels in both the serum and feces of OP mice were significantly higher than those of OR mice, and the body fat percentage of mice was positively correlated with folic acid contents in the serum and feces, confirming that systemic folic acid levels are closely associated with the obesity-susceptible phenotype. In vitro, folic acid markedly inhibited 3T3-L1 preadipocyte differentiation and intracellular lipid accumulation, and reduced triglyceride content in adipocytes. It significantly or highly significantly downregulated the expression of Pparγ, Acc1, Pparα, Fasn, Lpl, and Hsl, while highly significantly upregulating the expression of Cpt1α. Molecular docking revealed that the binding affinity between folic acid and PPARγ was ?25.52 kJ/mol, indicating favorable binding activity between the two molecules. In conclusion, folic acid significantly modulates the adipogenic differentiation of 3T3-L1 preadipocytes and acts as a key substance in lipid metabolism, playing an essential role in adipocyte differentiation and the regulation of obesity.