In parasites of the order Kinetoplastida, such as Trypanosoma cruzi and Trypanosoma brucei, glycolysis is carried out by glycolytic enzymes in glycosomes. One of the glycolytic enzymes is triosephosphate isomerase (TIM), which in T. brucei is localized exclusively in glycosomes, whereas in T. cruzi, the localization of TIM has not been fully ascertained. In the present work, we made a monoclonal antibody (mAb 6-11G) against recombinant T. cruzi TIM (rTcTIM). Incubation of T. cruzi epimastigotes with the mAb inhibited parasite survival. Western blotting showed that the mAb recognized rTcTIM and a 27 kDa band in T. cruzi lysates that corresponded to TcTIM. Sera from patients with Chagas disease recognized rTcTIM and cross-reacted with human recombinant TIM. The cross reactivity between parasite and human TIM possibly contributes to the autoimmune pathogenesis of Chagas disease. Electron microscopy of T. cruzi epimastigotes with the mAb showed that TIM was located within glycosomes, in the cytoplasm, the nucleus, and the kinetoplast. Collectively, the data shed new light on T. cruzi TIM and opens perspectives for drug design.