The Retinoid X Receptor (RXR) is an attractive target in the treatment of colon cancer. Different therapeutic binders with high potency have been used to specifically target RXR. Among these compounds is a novel analogue of berberine, B12. We provided structural and molecular insights into the therapeutic activity properties of B12 relative to its parent compound, berberine, using force field estimations and thermodynamic calculations. Upon binding of B12 to RXR, the high instability elicited by RXR was markedly reduced; similar observation was seen in the berberine-bound RXR. However, our analysis revealed that B12 could have a more stabilizing effect on RXR when compared to berberine. Interestingly, the mechanistic behaviour of B12 in the active site of RXR opposed its impact on RXR protein. This disparity could be due to the bond formation and breaking elicited between B12/berberine and the active site residues. We observed that B12 and berberine could induce a disparate conformational change in regions Gly250-Asp258 located on the His-RXRα/LBD domain. Comparatively, the high agonistic and activation potential reported for B12 compared to berberine might be due to its superior binding affinity as evidenced in the thermodynamic estimations. The total affinity for B12 (−25.76 kcal/mol) was contributed by electrostatic interactions from Glu243 and Glu239. Also, Arg371, which plays a crucial role in the activity of RXR, formed a strong hydrogen bond with B12; however, a weak interaction was elicited between Arg371 and berberine. Taken together, our study has shown the RXRα activating potential of B12, and findings from this study could provide a framework in the future design of RXRα binders specifically tailored in the selective treatment of colon cancer.