Leishmania infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63 and others) that promote survival inside the cells. Extracellular vehicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different Leishmania species to modulate inflammatory responses is scarce, especially from those species causing different clinical manifestations (visceral versus cutaneous). The purpose of this study was to expose murine macrophages and human peripheral blood mononuclear cells (PBMCs) to EVs from three Leishmania species from New World including L. infantum, L. braziliensis and L. amazonensis. Briefly, EVs were released from promastigote forms, purified by ultracentrifugation and quantitated by Nanoparticle Tracking Analysis (NTA) prior to murine macrophage exposure. EVs were also checked by Scanning Electron Microscopy (SEM). We used two types of cells including murine macrophages and PBMCs from human donors. For the murine peritoneal macrophage assays, we quantitated NO and cytokine production (TNF-, IL- 6 and IL-10) using CBA flex kit (BD Biociences). For PBMCs, we determined IL-32 production using ELISA. For NF-KB translocation, we performed assays in THP-1 cells and the fluorescence was detected using p65 antibody. NTA analysis did not show any differences in the EV sizes among the strains. EVs from L. braziliensis and L. infantum failed to induce a pro-inflammatory response. EVs from both L. infantum WT and LPG-deficient mutant (LPG-KO) did not show any differences in their interaction with murine macrophages, suggesting that LPG solely was not determinant for activation. However, in PBMCs, WT L. infantum/L. amazonensis EVs induced higher levels of IL-32 compared to (LPG-KO). On the other hand, in murine macrophages, EVs from L. amazonensis were immunomodulatory inducing NO, TNF-α, IL-6 and IL-10 via TLR4 and TLR2. To determine whether such activation was related to NF-B p65 translocation, THP-1 macrophage cells were exposed to EVs. In the same way, only EVs from L. amazonensis exhibited a highly percentage of cells positive for NF-B. Our results suggest an important role of EVs in determining the pattern of immune response depending on the parasite species and the host. For L. infantum, LPG deficient-EVs were not determinant for the activation of murine macrophages. However, this activation was very important to induce IL-32 in PBMCs. Depending on the host and Leishmania species, EVs may trigger a diverse immune activation. Those polymorphisms during this process may be determinant for the wide spectrum of the clinical manifestations that they cause.