Ecto-30-nucleotidase/nuclease (30NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 30mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-30-nucleotidase activity (La30-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La30-nucleotidase inhibitor and approach the possible involvement of ecto-30-nucleotidase in cellular adhesion. La30-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La30-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu2+ ions. Interestingly, ecto-30-nucleotidase activity is 60-fold higher than that of ecto-50-nucleotidase in L. amazonensis. Additionally, ecto-30-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage–parasite attachment/invasion was increased by 400% in the presence of adenosine 30-monophosphate (30AMP); however, this effect was reverted by TTM treatment. We believe that La30-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection