Coenzyme Q10 (CoQ10) is a well-known anti-adipogenic factor that possesses the capability to regulate non-alcoholic fatty liver disease (NAFLD). However, the mechanism by which CoQ10 acts on NAFLD is still unclear. In this study, the role of CoQ10 in the prevention of NAFLD was investigated in vivo and in vitro. C57BL/6J mice were fed a normal diet, high-fat diet (HFD) or HFD supplemented with CoQ10 (1800 mg kg⁻¹ HFD) for 24 weeks. HepG2 cells were treated with sodium palmitate for investigating the mechanism of action of CoQ10 on NAFLD. The results showed that CoQ10 alleviated HFD-induced weight gain and NAFLD, accompanied by an anti-hyperlipidaemia effect, by reducing the serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels. Importantly, CoQ10 could downregulate the expression of sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), which are related to lipid synthesis, and upregulate the expression of peroxisome proliferator-activated receptors α (PPARα) and carnitine palmitoyltransferase-1 (CPT-1) associated with fatty acid oxidation. Similar to the results from mice, treatment with CoQ10 alleviated sodium palmitate-induced hepatocyte steatosis via the inhibition of lipogenesis and promotion of fatty acid oxidation. However, Compound C, as an AMPK inhibitor, could significantly block the benefits derived from CoQ10 treatment. In conclusion, CoQ10 could serve as an AMPK activator and regulate the hepatic lipid metabolism to inhibit the abnormal accumulation of hepatic lipids and prevent NAFLD progression.