The trehalose analogue compound validamycin is a very strong competitive inhibitor of trehalase enzymatic activity in various insects, and it has been studied as a potential material for the development of new insecticides. The inhibitory effect of validamycin on trehalase activity in the red flour beetle, Tribolium castaneum Herbst, was investigated in the present study. We measured changes in glucose concentrations and assessed morphological abnormalities in the wings of T. castaneum after trehalase activity was inhibited by validamycin treatment. We found that the application of validamycin during the larval stage, via both feeding and injection methods, resulted in a significant decrease in trehalase activity during the larval, pupal, and adult stages. Injection of validamycin into pupae caused a reduction in trehalase enzyme activity and caused the glucose concentration to fall by 21% compared with the control. The dose-dependent lethal effect of validamycin treatment was recorded, with the highest mortality rate of ∼78%. This study is the first to demonstrate the dosedependent effect of validamycin on the wing morphology of T. castaneum adults, in that the injected insects exhibited wrinkled elytra and improperly folded hindwings. Reduction of elytral length and width was also recorded. The trehalase inhibitor validamycin is valuable for investigating the physiological function of trehalase in insects and may be useful as a pest control tool for T. castaneum.