Chagas disease is a neglected disease, caused by the protozoan Trypanosoma cruzi. This kinetoplastid presents a cycle involving different forms and hosts, being trypomastigotes the main infective form. Despite various T. cruzi proteomic studies, the assessment of bloodstream trypomastigote profile remains unexplored. The aim of this work is T. cruzi bloodstream form proteomic description. Employing shotgun approach, 17,394 peptides were identified, corresponding to 7514 proteins of which 5901 belong to T. cruzi. Cytoskeletal proteins, chaperones, bioenergetics-related enzymes, and trans-sialidases are among the top-scoring. GO analysis revealed that all T. cruzi compartments were assessed; and majority of proteins are involved in metabolic processes and/or presented catalytic activity. The comparative analysis between the bloodstream trypomastigotes and cultured-derived or metacyclic trypomastigote proteomic profiles pointed to 2202 proteins exclusively detected in the bloodstream form. These exclusive proteins are related to: (a) surface proteins; (b) non-classical secretion pathway; (c) cytoskeletal dynamics; (d) cell cycle and transcription; (e) proteolysis; (f) redox metabolism; (g) biosynthetic pathways; (h) bioenergetics; (i) protein folding; (j) cell signaling; (k) vesicular traffic; (l) DNA repair; and (m) cell death. This large-scale evaluation of bloodstream trypomastigotes, responsible for the parasite dissemination in the patient, marks a step forward in the comprehension of Chagas disease pathogenesis. CONCLUSIONS The hemoflagellate protozoan T. cruzi is the etiological agent of Chagas disease and affects people by the millions in Latin America and other non-endemic countries. The absence of efficient drugs, especially for treatment during the chronic phase of the disease, stimulates the continuous search for novel molecular targets. The identification of essential molecules, particularly those found in clinically relevant forms of the parasite, could be crucial. Inside the vertebrate host, trypomastigotes circulate in the bloodstream before infecting various tissues. The exposure of bloodstream forms of the parasite to the host immune system likely leads to differential protein expression in the parasite. In this context, an extensive characterization of the proteomic profile of bloodstream trypomastigotes could help to find not only promising drug targets but also antigens for vaccines or diagnostics. This work is a large-scale proteomic assessment of bloodstream trypomastigotes that show a considerable number of proteins belonging to different metabolic pathways and functions exclusive to this parasitic form, and provides a valuable dataset for the biological understanding of this clinically relevant form of T. cruzi.