Retinoic X receptor (RXR) is a promising target for drug discovery against cancer and metabolic syndromes. Here, we identified a specific RXRα antagonist, danthron, from the traditional Chinese medicine rhubarb. Danthron repressed all tested RXRα-involved response element transcription, including the RXRE, PPRE, FXRE, and LXRE. Results from native PAGE and isothermal titration calorimetry (ITC)-based assays indicated that danthron bound to the tetrameric RXRα-LBD in a specific stoichimetric ratio, and such a binding could influence the corepressor SMRT affinity to the receptor. Additionally, a unique tetrameric structure of the apo-RXRα ligand-binding domain (LBD) was determined, which exhibited a larger tetramer interface and different ligand-binding pocket size compared with the one previously reported. Together with the biochemical and biophysical results, the determined crystal structure of danthron-soaked RXRα-LBD suggested a new mechanism for danthron antagonism to tetrameric RXRα. Moreover, the in vivo efficient improvement of insulin sensitivity by danthron was observed in diet-induced obese (DIO) mice. Thus, our findings were expected to supply new insights into the structural basis of RXRα antagonist for its further potential therapeutic application.