Updated project metadata. Trypanosoma cruzi, the etiological agent of Chagas disease, has a complex life cycle occurring between triatomine insects (responsible for the vectorial transmission of the infection) and mammals. Inside the insect vector, epimastigote forms (proliferative, non-infective to mammals) differentiate into metacyclic trypomastigotes forms (non-proliferative, infective to mammals). This differentiation process happens at the final portion of insect vector’s gut and it is crucial for the progress of T. cruzi life cycle. Factors that play a role in metacyclogenesis include temperature, pH and nutritional stress followed by availability of specific metabolites. Previous results showed that some amino acids such as Pro, His and Gln are able to fully support cell differentiation whereas the BCAAs completely block it. In addition, combination of the “pro-metacyclogenic” amino acid Pro with one of the “anti-metacyclogenic” BCAAs produces viable metacyclics, however, with a drastically reduced infectivity. In order to investigate the peculiarities of metacyclic parasites differentiated in the presence of BCAAs, we performed proteomics analyses to compare metacyclics obtained in triatomine artificial urine (TAU) supplemented with Pro alone and in combination with Leu, Ile or Val. Our analyses revealed that when compared to the metacyclics originated in TAU-Pro, 40 proteins are differentially regulated in metacyclics from TAU-Pro-Leu, whereas 131 and 179 are differentially regulated in TAU-Pro-Ile and TAU-Pro-Val, respectively. From each group, 22, 11 and 13% of the proteins are putatively related to metabolic processes, respectively. Overall, enzymes involved in both glycolysis and TCA cycle are reduced in metacyclics obtained in the presence of Pro-BCAAs whereas enzymes involved in amino acid and purine metabolic pathways are increased. In addition, metacyclics obtained in the presence of Pro-Ile and Pro-Val have enzymes involved in lipid and redox metabolism increased. Together our analyses indicate that metacyclics obtained in the presence of BCAA can have their metabolism reshaped, which in turn can contribute to the reduced infectivity observed in these parasites.