The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is primarily driven by excessive lipid accumulation and metabolic dysregulation, necessitating a comprehensive investigation into the underlying mechanisms. This study employed an in vitro model, wherein Huh7 cells were induced with a palmitic acid/oleic acid mixture, and an in vivo model involving the provision of a high-fat diet to SD rats for six weeks. Employing techniques such as oil red O staining, immunofluorescence, and Western blotting, we examined lipid synthesis, metabolism, and the associated molecular pathways. The findings indicate that GPR75 overexpression markedly enhances lipid synthesis and impairs lipid metabolism. Conversely, GPR75 knockdown significantly diminished the fluorescence intensity of lipid synthesis factors FASN and SREBP1, concurrently elevating the expression of AMPK and SIRT1 proteins, which culminated in reduced lipid synthesis and improved lipid metabolism. Furthermore, inhibiting the AMPK-SIRT1 pathway following GPR75 knockdown led to a significant reversal of these lipid metabolic alterations. Overall, our study elucidates that GPR75 inhibition may diminish lipid accumulation and enhance lipid metabolism both in vitro and in vivo, primarily through the activation of the AMPK-SIRT1 signaling pathway.