The characterization of the structure of mumbaistatin (1), an effective inhibitor of the glucose-6-phosphatase system (EC 3.1.3.9), is reported. Isolation of mumbaistatin from cultures of Streptomyces sp. DSM 11641 was achieved by anion-exchange and reversed-phase chromatography. The acid-labile inhibitor was methylated for the structure determination. Single-crystal X-ray structure analysis of a triply methylated dehydration product, C31H24O11, revealed the structure of an aromatic dispirodiketal (2), a compound containing a previously undescribed ring system. Extensive 2D-NMR experiments with mumbaistatin and with the methylation products showed that mumbaistatin itself possesses the hydroxydiketodicarboxylic acid structure 1, C28H20O12, which, in the presence of acid or upon activation through methyl ester formation, undergoes self-condensation with loss of water to the dispirodiketal form (2). Mumbaistatin is an anthraquinone derivative, whose open-chain diketo form acts as a specific and powerful inhibitor of glucose-6-phosphate translocase: IC50=5 nM. The activity towards the same enzyme of the cyclized dispirodiketal derivatives is roughly one thousand times lower.