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.