Rhinacanthin C (RC) is a naphthoquinone ester with an anti‐inflammatory activity extracted from Rhinacanthus nasutus (L.) Kurz (Rn). It has been proven to improve hyperglycemia and hyperlipidemia, but the prevention and mechanism of RC in nonalcoholic fatty liver disease (NAFLD) are not clear. In the current study, we first extracted RC from Rn using ethyl acetate and identified it by HPLC, MS, and NMR. At the same time, molecular docking analysis of RC with AMPK and SREBP‐1c was performed using AutoDock software. In addition, the mouse model of NAFLD was induced by a high‐fat diet in vivo, and low, medium, and high concentrations of RC were used for intervention. The results showed that RC significantly reduced the body mass and liver body coefficient of NAFLD mice, inhibited liver inflammation and fat accumulation, and improved insulin resistance. Further studies showed that RC significantly reduced the levels of serum leptin and resistin, upregulated the expression levels of adiponectin and adiponectin receptor in the liver, and inhibited the expression levels of MCP‐1, TNF‐α, and IL‐6. In terms of mechanism, RC upregulates the expression of p‐AMPK and SIRT1 and downregulates the expression of p‐p65, SREBP‐1c, Fas, Acc‐α, PPAR‐γ, and SCD1. These studies suggest that RC improves insulin resistance and lipid accumulation in NAFLD by activating the AMPK/SIRT1 and SREBP‐1c/Fas/ACC pathways, respectively.