A structural study of trehalostatin 1, a specific inhibitor of blowfly trehalase, revealed that 1 contains an unusual five-membered pseudocyclitol. Trehalostatin 1, a potent and specific inhibitor against blowfly (Aldrichna grahami) trehalase was purified from the culture broth of Amycolatopsis trehalostatical as a white amorphous powder: [α] +115° (c. 1.0, H2O), IC50 0.68 ng ml-1 (for blowfly trehalase). Several colour reactions provided evidence that 1 contains a saccharide moiety and a C-N linkage, but neither a reducing terminal nor a free amino group. IR (1610 cm-1 absorption) and UV endo-absorption suggested a saccharide with amide or ureido functions. Liquid SIMS spectrum gave [M + H]+ at m/z 367, indicating molecular formula C13H22O10N2. Intensive NMR studies of its acetates (2 and 3) revealed three proton spin systems: an α-glucopyranosyl residue and fragments A/B, leading to the structure of trehalostatin 1 with a glycosyl isoureido functional group that can tautomerize (showing acid/base properties). Compared to known trehalase inhibitors with basic imine functions, trehalostatin 1's high selectivity for insect trehalase is probably due to the ambident isoureido structure, which neutralizes the carboxylic acid/carboxylate 'flip-flop' system at the insect trehalase active site.