The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with β-trypsin was solved and refined at 1.55 Å to a crystallographic R-factor of 14.7% and R_free of 16.2%. The BTCI exhibits low average overall B-factors compared with other proteinase inhibitors. The BTCI folds into two compact subdomains of similar tertiary structure to those of BBI's with closest conformation in the reactive site loops. An unusual buried cluster of charged side-chains, located in the inter-subdomain region, and the high conserved aromatic cluster in a typical orthogonal geometry have been identified. In the complex, the recognition is made mainly by inhibitor's Lys26 that is fully embedded into the S1 pocket of trypsin, forming a buried salt-bridge with the Asp189, mediated by a water molecule. This protein-protein interface is stabilized by hydrophobic contacts and an extensive hydrogen bond network, with the participation of the two water molecules and a PEG molecule.