Monoxenous trypanosomatids usually have an invertebrate as the only host in their life cycles, however, they have been found repeatedly in plants and/or mammals. To succeed in colonizing a vertebrate host, the parasite must quickly adapt to drastic changes in the environment (e.g. temperature), which reflect the conditions found in the insect and mammalian hosts. Leishmanolysin is a metalloprotease ubiquitously distributed in trypanosomatids, playing a myriad of functions. In Herpetomonas samuelpessoai, an insect trypanosomatid, the leishmanolysin-like molecule was implicated in the nutrition and insect adhesion. Herein, we showed that leishmanolysin expression is equally expressed in H. samuelpessoai parasites submitted to insect (26 °C) and mammalian (37 °C) temperatures. Also, the parasites grown in both temperatures interacted at similar rates with macrophages. Finally, we showed that leishmanolysin is involved in crucial steps in the interaction of H. samuelpessoai cells with macrophages, since the treatment with either anti-leishmanolysin antibodies or metalloprotease inhibitor 1,10-phenanthroline significantly reduced the association index. Similarly, the treatment of the macrophages with purified leishmanolysin promoted a powerful reduction in the association index, suggesting the direct involvement of macrophage receptors. These results suggest that H. samuelpessoai leishmanolysin molecules are not modulated by temperature and are involved in the interaction with mammalian cells.