Background: Trypanothione reductase (TR) helps to maintain an intracellular reducing environment in trypanosomatids, a group of protozoan parasites that afflict humans and livestock in tropical areas. This protective function is achieved via reduction of polyamine-glutathione conjugates, in particular trypanothione. TR has been validated as a chemotherapeutic target by molecular genetics methods. To assist the development of new therapeutics, we have characterized the structure of TR from the pathogen Trypanosoma cruzi complexed with the substrate trypanothione and have used the structure to guide database searches and molecular modelling studies. Results: The TR-trypanothione-disulfide structure has been determined to 2.4 Å resolution. The chemical interactions involved in enzyme recognition and binding of substrate can be inferred from this structure. Comparisons with the related mammalian enzyme, glutathione reductase, explain why each enzyme is so specific for its own substrate. A CH···O hydrogen bond can occur between the active-site histidine and a carbonyl of the substrate. This interaction contributes to enzyme specificity and mechanism by producing an electronic induced fit when substrate binds. Database searches and molecular modelling using the substrate as a template and the active site as receptor have identified a class of cyclic-polyamine natural products that are novel TR inhibitors. Conclusions; The structure of the TR-trypanothione enzyme-substrate complex provides details of a potentially valuable drug target. This information has helped to identify a new class of enzyme inhibitors as novel lead compounds worthy of further development in the search for improved medicines to treat a range of parasitic infections.