BACKGROUND: Extracellular vesicles (EVs) are structures with phospholipid bilayer membranes and 100-1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles. METHODOLOGY/PRINCIPAL FINDINGS: In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50-300 nm-diameter extracellular vesicles (EVs). The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1ß, and TNF-α. CONCLUSIONS/SIGNIFICANCE: EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1ß and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.