Leishmaniasis is a group of infectious diseases caused by different species of Leishmania, which affects about 2 million people per year. Although Leishmania is hypoendemic in most of the 88 endemic countries, up to 40% of the population in these countries produces a Leishmania-positive skin test in rural endemic areas, illustrating that contact with the parasite is much more widespread than previously expected. Several calpain inhibitors are under development and some are useful against important human pathogens. In this study, we report the effect of MDL28170, a potent calpain inhibitor, on the growth and morphology of Leishmania amazonensis. Briefly, promastigotes were counted using a Neubauer chamber and resuspended in fresh medium to a final concentration of 1.0 “ 10⁶ viable promastigotes per milliliter. The inhibitor was added to the culture at final concentrations of 15, 20, 25 and 30 mM. The calpain inhibitor at 30 mM promoted a powerful reduction on the cellular growth rate by approximately 38, 90, 94 and 95% after 24, 48, 72 and 96 h, respectively. The lowest concentrations of the drug (15 and 20 mM) presented significant inhibitory effects only after 72-96 h of growth. Corroborating these results, optical microscopy observations showed a massive deterioration of promastigote cells after treatment of the parasites with 30 mM MDL 28170 for 48 h. Based on the effects of MDL28170 on the growth rate and morphology of L. amazonensis, we aimed to detect calpain homologues in this protozoan by immunoblot assays using different anti-calpain antibodies. The antibodies against Drosophila melanogaster calpain (Dm-calpain) strongly recognized a polypeptide band migrating at approximately 80 kDa. No common epitopes were found between mammalian calpains and L. amazonensis polypeptides. The calpain-like molecule was detected on the cell surface of L. amazonensis, as demonstrated by flow cytometry and fluorescence microscopy analyses using the anti-Dm-calpain antibody. The immunofluorescence image showed a labeling throughout the cell surface, including the flagellum.These results add new in vitro insights into the exploitation of calpain inhibitors in treating parasitic infections and add this family of proteases to the list of potential targets for development of more potent and specific inhibitors against trypanosomatids.