Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are targets for the development of new anti-parasitic therapy. Protozoan parasites like Leishmania predominantly express Clan CA cysteine proteases for key life cycle functions. It was therefore unexpected to find a high level of serine protease activity expressed by Leishmania donovani. Purification of this activity followed by mass spectrometry identified oligopeptidase B (OPB; Clan SC, family S9A) as the responsible enzyme. This was confirmed by gene knock-out of OPB, which resulted in the disappearance of the detected serine protease activity of Leishmania extracts. To delineate the specific role of OPB in parasite physiology, proteomic analysis was carried out on OPB(-/-) versus wild type parasites. Four protein species were significantly elevated in OPB(-/-) parasites, and all four were identified by mass spectrometry as enolase. This increased enolase was enzymatically inactive and associated with the parasite membrane. Aside from its classic role in carbohydrate metabolism, enolase was recently found to localize to membranes, where it binds host plasminogen and functions as a virulence factor for several pathogens. As expected, there was a striking alteration in macrophage responses to Leishmania when OPB was deleted. Whereas wild type parasites elicited little, if any, response from infected macrophages, OPB(-/-) parasites induced a massive up-regulation in gene transcription. Additionally, these OPB(-/-) parasites displayed decreased virulence in the murine footpad infection model.