Danthron (0.1, 1, and 10 μM) dose-dependently promotes the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in both HepG2 and C2C12 cells. Meanwhile, Danthron treatment significantly reduces the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions, and the total cholesterol (TC) and triglyceride (TG) levels. In addition, Danthron treatment efficiently increases glucose consumption. Danthron effectively reduces intracellular lipid contents and enhances glucose consumption in vitro via activation of AMPK signaling pathway. 10 μM Danthron/24 h is safe for HepG2 cells. With 80% confluence, HepG2 cells are incubated with Danthron (0.1-10 μM) in FBS-Free media for 8 h. Subsequently, cells are harvested for Western blot assay. Danthron increases the p-AMPK protein in a dose-dependent manner, and no changes in t-AMPK protein are observed. Danthron inhibits 9-cis retinoic acid (9cRA)-induced retinoic X receptor α (RXRα) transactivation by IC50 at 0.11 μM. To further clarify the stoichimetric ratio of Danthron binding to RXRα-ligand-binding domain (LBD), isothermal titration calorimetry (ITC) experiment is performed. The KD value of Danthron binds to RXRα-LBD by ITC experiment is determined at 7.5 μM.