Reducing fat deposits in hepatocytes is a direct treatment for nonalcoholic fatty liver disease (NAFLD) and the fatty acid metabolic processes mediated by fatty acid β-oxidation are important for the prevention of NAFLD. In this study, we established high-fat-diet models in vitro and in vivo to investigate the mechanism by which hesperidin (HDN) prevents NAFLD by modulating fatty acid β oxidation. Based on LC-MS screening of differential metabolites, many metabolites involved in phospholipid and lipid metabolism were found to be significantly altered and closely associated with fatty acid β-oxidation. The results from COIP experiments indicated that HDN increased the deacetylation of PGC1α by SIRT1. In addition, the results of CETSA and molecular docking experiments suggest that HDN targeting of SIRT1 plays an important role in their stable binding. Meanwhile, it was found that HDN reduced fatty acid uptake and synthesis and promoted the expression of SIRT1/PGC1α and fatty acid β-oxidation, and the latter process was inhibited after transfection to knockdown SIRT1. The results suggest that HDN improves NAFLD by promoting fatty acid β-oxidation through activating SIRT1/PGC1α. Thus, the findings indicate that HDN may be a potential drug for the treatment of NAFLD.