The high-fat diet (HFD) has dramatically increased the prevalence of non-alcoholic fatty liver disease (NAFLD), such that it is the leading cause of chronic liver disease worldwide. With novel knowledge on pathogenesis of NAFLD, we illustrated molecular targets for pharmacologic and experimental approaches. Our study showed that quercetin was the valuable drug candidate, and the core targets such as PPARα, MCAD, LCAD, CPT1-L, CPT2 and FATP2 which participate in fatty acid oxidation were selected to perform further in vivo experiments based on network pharmacology and molecular docking technology. The effect was investigated in a high-fat diet-induced NAFLD male Sprague Dawley rat model by hepatic biochemical assays. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transferase analyses assessed liver function. Liver tissues were collected for histological staining, quantitative real-time PCR and western blotting. Greatly, quercetin reduced hepatic lipid accumulation and inflammation, ameliorated pathological liver changes, and up-regulated the expression levels of hepatic effectors in fatty acid oxidation. The results suggest the potential of quercetin as a nutritional supplement to HFD-induced NAFLD. Furthermore, the antagonist and agonist of PPARα provided a reliable scientific basis that these above-mentioned effectors were activated by quercetin via the PPAR pathway.