Antimycin A, a natural antibiotic from Streptomyces, specifically inhibits the electron transfer activity of ubiquinol-cytochrome c oxidoreductase (collectively called the bc1 complex) in most respiratory and photosynthetic organisms and is widely used in functional studies of the enzyme due to its high specificity and strong affinity. Recent progress in determining the three-dimensional structure of the eukaryotic bc1 complex revealed two separate cavities in the cytochrome b subunit, one binding inhibitors of quinone oxidation and the other (e.g., antimycin A) inhibitors of quinone reduction. However, analyzing specific interactions between the inhibitor's functional groups and the protein is difficult at the currently available ~3 Å resolution without knowing the exact geometry of the molecules involved. To facilitate structural analysis, we determined the three-dimensional structure of isolated antimycin A1 by X-ray crystallography and employed two-dimensional nuclear magnetic resonance (NMR) spectroscopy to resolve discrepancies between the X-ray structure and chemical structure. The crystal structure of antimycin A1 revealed two ring moieties—a planar 3-formylamino salicylic acid (FASA) and a puckered dilactone—connected via an amide bond, and the absolute configuration of the dilactone ring was determined. Surprisingly, electron density for the acyl side chain indicated a 1-methyl butanoate instead of the previously described 2-methyl isomer; NMR confirmed antimycin A1 consists of two isomers (4:1 ratio, with 1-methyl butanoate preferentially crystallizing). To determine the inhibitory mechanism, we grew cocrystals of the mitochondrial bc1 complex with the inhibitor and analyzed them by X-ray crystallography. The difference Fourier map showed the inhibitor binds near the high potential b heme, with density matching antimycin A's crystal structure. Cocrystal density provided insights into binding: polar residues interact with the FASA ring, while nonpolar residues form a hydrophobic environment for the dilactone ring. We conclude antimycin A1 inhibits electron transfer by displacing ubiquinone from the Qi pocket of cytochrome b. This structural knowledge will aid further refinement of the bc1 complex structure and the design of more potent insecticides/herbicides targeting specific species' bc1 complexes.