We report the functional characterization in Leishmania amazonensis of a soluble pyrophosphatase (LaVSP1) which localizes in acidocalcisomes, a vesicular acidic compartment. LaVSP1 is preferentially expressed in metacyclic forms. Experiments with dominant negative mutants show the requirement of LaVSP1 functional expression for metacyclogenesis and virulence in mice. Depending on the pH and the cofactors Mg2+ or Zn2+, both present in acidocalcisomes, LaVSP1 hydrolyzes either inorganic pyrophosphate (Km=92 µM, kcat=125 s-1), tripolyphosphate (Km=1153 µM, kcat=131 s-1) or polyphosphate of 28 residues (Km=123 µM, kcat=8 s-1) . Predicted structural analysis suggests that the structural orientation of the residue K78 in LaVSP1 accounts for the observed increase in Km compared with the yeast pyrophosphatase and for the ability of trypanosomatid VSP1 enzymes to hydrolyze polyphosphate. These results make the VSP1 enzyme an attractive drug target against trypanosomatid parasites.