Combretastatin A4 (CA-4) derivatives are microtubule-destabilizing agents, some of which are in advanced clinical trials for cancer therapy. The active cis conformation of CA-4 can readily isomerize into a thermodynamically more stable but significantly less active trans form. Here, we solved the high-resolution crystal structure of cis-CA-4 in complex with tubulin. The compound binds to the colchicine site of tubulin and displays both common and distinct interaction points with colchicine. Using metadynamics simulations, we generated the trans form of the ligand within its binding site and computed the relative binding free energy of the cis-CA-4 and trans-CA-4 isomers via a thermodynamic cycle. The calculations suggest structural distortions of the bound trans-CA-4 molecule as the likely cause of its reduced activity in comparison with that of its cis isomer. Our findings could open up unique possibilities for structure-guided drug engineering with the aim of discovering combretastatin variants with improved chemical properties and pharmacological profiles.