Abstract Compound cordycepin (3?-deoxyadenosine) has been used in cancer treatment, diabetes prevention, and anti-virus, as well as hyperlipidemia downregulation in vivo. However, the association between lipid metabolism and mitochondrial activity in cordycepin-treated hepatocytes is remain unclear. In this study, whether cordycepin affects lipogenesis and fatty beta-oxidation through regulating mitochondrial activity and AMP-activated protein kinase (AMPK) in hepatocytes were investigated. The data showed that cordycepin decreased lipid accumulation via activating AMPK and regulating mitochondrial function in oleic acid (OA)-induced mouse FL83B hepatocytes while the anti-fatty liver effect of cordycepin on regulating lipogenesis and fatty beta-oxidation was abolished by AMPK inhibitor compound C treatment. These results suggested that cordycepin attenuated lipid accumulation through promoting beta-oxidation and may be developed as a functional food/natural medicine for fatty liver suppression. Practical applications Traditional herb, Chinese medicine, and functional food treatments for hyperlipidemia are relatively cheap and locally available. The results of this study suggested that cordycepin (active compound of Cordyceps) may be developed as functional foods for fatty liver treatment via regulating fatty beta-oxidation.