Background and Aims: Lipid accumulation is the major characteristic of non-alcoholic fatty liver disease, the preva-lence of which continues to rise. We aimed to investigate the effects and mechanisms of icaritin on lipid accumula-tion. Methods: Cells were treated with icaritin at 0.7, 2.2, 6.7, or 20 μM for 24 h. The effects on lipid accumulation in L02 and Huh-7 cells were detected by Bodipy and oil red O staining, respectively. Mitochondria biogenesis of L02 cells was detected by MitoTracker Orange staining. Glucose up-take and adenosine triphosphate content of 3T3-L1 adipo-cytes and C2C12 myotubes were detected. The expression levels of proteins in the adenosine 5′-monophosphate-acti-vated protein kinase (AMPK) signaling pathway, biomarkers of autophagy, and mitochondria biogenesis were measured by western blotting. LC3 puncta were detected by immu-nofluorescence. Results: Icaritin significantly attenuated lipid accumulation in L02 and Huh-7 cells and boosted the mitochondria biogenesis of L02 cells. Icaritin enhanced glu-cose uptake, decreased adenosine triphosphate content, and activated the AMPK signaling pathway in 3T3-L1 adipo-cytes and C2C12 myotubes. Icaritin boosted autophagy and also enhanced the initiation of autophagic flux in 3T3-L1 preadipocytes and C2C12 myoblasts. However, icaritin de-creased autophagy and promoted mitochondria biogenesis in 3T3-L1 adipocytes and C2C12 myotubes. Conclusions: Icaritin attenuates lipid accumulation by increasing energy expenditure and regulating autophagy by activating the AMPK pathway.