Cysteine proteases (CPs) of the papain superfamily expressed by pathogenic protozoa have been implicated in their survival and interaction with the mammalian host. Potent synthetic irreversible inhibitors of T. cruzi CP, cruzipain, have been successfully tested in animal models of T. cruzi infection. Such inhibitors are also effective in preventing the in vitro survival of Trypanosoma b. brucei, a pathogenic protozoa used in laboratories as a model to study Human Sleeping Sickness. However, the primary target of these drugs in T. brucei, brucipain, has not been characterized in detail. We have previously reported the expression of functional brucipain and the preliminary characterization of its biochemical properties. Here, we studied the substrate specificity of brucipain using synthetic 7-mer peptidyl substrates containing systematic substitutions in the P₁, P₂ and P₁' positions. We also determined the inactivation constants for the inhibition of brucipain by different natural and synthetic inhibitors of Clan CA proteases. We found that brucipain shows preference for small hydrophobic residues at the P₂ position and bulky hydrophobic residues in the P₁ position. Unlike cruzipain, that presents dual cathespin L and cathepsin B-like specificity, brucipain does not hydrolyze substrates containing Arg at the P₂ position, showing strict cathepsin L-like specificity. Brucipain is not capable of hydrolyzing substrates presenting Pro at P₁' position and shows preference for Leu at this position. Brucipain was strongly inactivated by its chagasin-like endogenous inhibitor, TbICP (K_i 4 pM), human cystatin C (K_i 5 pM) and by the recombinant pro domain (10 kDa) of cruzipain (K_i16 pM). A synthetic peptidyl vinylsulphone inhibitor containing Leu in P₂ was 100-fold more efficient in inhibiting brucipain than that containing Phe at P₂. These results show that brucipain differs substantially from cruzipain regarding the S₂ specificity and that potent synthetic inhibitors to cruzipain are less effective in inhibiting brucipain.  The investigation of the structural basis for the substrate specificity of brucipain may aid in the design of drugs tailored for efficient inhibition of this T.brucei cysteine protease.