The enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi (TcG6PDH) catalyses the first step of the pentose phosphate pathway and is considered a promising target for the discovery of a new drug against Chagas Diseases. In the present work, we describe the crystal structure of TcG6PDH obtained in a ternary complex with the substrate glucose-6-phosphate and the reduced 'catalytic' cofactor NADPH, which reveals the molecular basis of substrate and cofactor recognition. A comparison with the homologous human protein sheds light on differences in the cofactor-binding site that might be explored towards the design of new NADP(+) competitive inhibitors targeting the parasite enzyme. This article is protected by copyright. All rights reserved.