We previously demonstrated that intradermal and intramuscular vaccination with Leishmania amazonensis promastigote antigens (LaAg) increases the susceptibility of BALB/c mice to cutaneous leishmaniasis. In this study, we investigated the role played by serine and cysteine proteases as disease-promoting components of LaAg. Mice were immunized by the intramuscular route with LaAg that was pre-treated with a pool of serine or cysteine protease inhibitors (SPi and CPi, respectively) prior to infection with L. amazonensis. Neutralization of either enzyme type reversed the disease-promoting effect of LaAg, as seen by the slower lesion development. However, the parasite burden was only effectively controlled in mice receiving SPi-treated LaAg. Protection was associated with diminished production of TGF-beta and particularly IL-10 in response to parasite antigens by the lesion-draining lymph node cells of vaccinated mice relative to control. In vitro, soluble proteases isolated from LaAg (LaSP-Sol) directly activated IL-4, IL-10 and TGF-beta production by immune cells. Like native LaAg, vaccination with LaSP-Sol primed mice to respond to parasite challenge with a strong Jones-Mote cutaneous hypersensitivity reaction, and increased susceptibility to infection. Furthermore, neutralization of serine but not cysteine proteases blocked the capacity of LaAg to sensitize mice for Jones-Mote reaction. Together, these results indicate that soluble serine proteases are key components of LaAg responsible for its disease-promoting immunity.