Protozoan parasites are responsible of important healthy problems, among others malaria, leishmaniasis and trypanosomiasis. The present work reports the characterization of the first mammalian ATP-binding cassette transporter, subfamily A (ABCA)-like in Trypanosoma cruzi. TcABC1 is a single copy gene differentially expressed along the life cycle of the parasite, being absent in its infective form. TcABC1 localizes to the plasma membrane, flagellar pocket and intracellular vesicles. Functional studies of TcABC1 in transfected parasites suggest that the protein is implicated in intracellular trafficking, as determined by the analysis of endocytosis and exocytosis events. The accumulation of the endocytic markers FM4-64 and NBD-SM is increased in transfected parasites. Similarly, ectophosphatase and ectoATPase activities are increased in TcABC1 overproducers. Indeed, transmission electronic microscopy analysis showed a higher number of intracellular vesicles in TcABC1 transfectants. Taken together, these results suggest that the protein is involved in the endocytic and exocytic pathways of T. cruzi.