Non-alcoholic fatty liver disease (NAFLD) has been a leading cause of chronic metabolic disease, seriously posing healthy burdens to the public, whereas interventions available for it are limited to date. Patchouli oil had been reported to attenuate hepatic steatosis in our previous study. β-patchoulene (β-PAE) is a representative component separated from patchouli oil with multiple activities, but its effect against NAFLD is still unknown. To investigate the effect and potential mechanism of β-PAE on NAFLD, we used high fat diet (HFD) in vivo and free fatty acid (FFA) in vitro to induce hepatic steatosis in rats and L02 cells, respectively. Histological examination was evaluated via Hematoxylin-eosin and oil red O staining. The parameters for hepatic steatosis were estimated via biochemical kits, western blotting and quantitative real-time PCR. Compound C, the inhibitor of AMPK, was applied further to examine the precise mechanism of β-PAE on NAFLD. Our results indicated that β-PAE significantly attenuated HFD-induced weight gain, hepatic injury, lipid deposition in serum and hepatic tissue as well as FFA induced-lipid accumulation. Besides, β-PAE markedly improved the expression of AMP-activated protein kinase (AMPK) and its downstream factors which correlate with hepatic lipid synthesis and oxidation in vivo and in vitro. Nevertheless, Compound C abrogated the benefits derived from β-PAE in L02 cells. In conclusion, these results suggest that β-PAE exerts AMPK agonist-like effect to regulate hepatic lipid synthesis and oxidation, eventually prevent NAFLD progression.