Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.