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This work is aimed at characterizing and evaluating the protective role of EVs released by Leishmania amazonensis promastigotes under different maintenance conditions. Initially, using a control strain, we standardized 26\u00b0C as the best release temperature to obtain EVs with a potential protective role in the experimental leishmaniasis model. Then, long-term (LT-P) promastigotes of L. amazonensis were obtained after long-term in vitro culture (100 in vitro passages). In vivo-derived (IVD-P) promastigotes of L. amazonensis were selected after 3 consecutive experimental infections in BALB\/c mice. Those strains developed similar lesion sizes except for IVD-P at 8 weeks post infection. No differences in EV production were detected in both strains. However, the presence of LPG between LT-P and IVD-P EVs was different. Groups of mice immunized with EVs emulsified in the adjuvant and challenged with IVD-P parasites showed decreased lesion size and parasitic load compared with the nonimmunized groups. The immunization regimen with two doses showed high IFN-\u03b3 and IgG2a titers in challenged mice with either IVD-P or LT-P EVs. IL-4 and IL-10 were detected in immunized mice, suggesting a mixed Th1\/Th2 profile. EVs released by either IVD-P or LT-P induced a partial protective effect in an immunization model. Thus, our results uncover a potential protective role of EVs from L. amazonensis for cutaneous leishmaniasis. 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