Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the ordersDipteraandLepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and the suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquitoAnopheles gambiaeis a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity of these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Aminoacid residue Asp111 in Nobo is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.