Tetronothiodin (1) is a potent and selective cholecystokinin type B (CCK-B) receptor antagonist produced by Streptomyces sp. NR0489. Its structure was elucidated to be a macrocyclic compound comprising cyclohexene, alpha-acyltetronic acid and tetrahydrothiophene moieties based on various 2D NMR experiments on 1 and its dihydro derivative. The stereochemistries for the cyclohexene and tetrahydrothiophene rings were elucidated based on the analysis of NOEs obtained by NOESY experiments and NOE difference spectroscopy. The relative configuration of the cyclohexene moiety in 1 was revealed to be the same as that of the corresponding part in kijanimicin and chlorothricin, which can be structurally related to 1 in terms of their containing a cyclohexene ring with a spirotetronic acid in the molecule.