Bowman–Birk inhibitors (BBI) from legumes, such as soyabean, pea, lentil and chickpea, are naturally occurring plant protease inhibitors which have potential health-promoting properties within the mammalian gastrointestinal tract. BBI can survive both acidic conditions and the action of proteolytic enzymes within the stomach and small intestine, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. In a previous study, we reported the ability of a recombinant form of TI1B (rTI1B), representing a major BBI isoinhibitor from pea, to influence negatively the growth of human colorectal adenocarcinoma HT29 cells in vitro. In the present study, we investigate if this effect is related directly to the intrinsic ability of BBI to inhibit serine proteases. rTI1B and a novel engineered mutant, having amino acid substitutions at the P1 positions in the two inhibitory domains, were expressed in the yeast Pichia pastoris. The rTI1B proved to be active against trypsin and chymotrypsin, showing Ki values at nanomolar concentrations, whereas the related mutant protein was inactive against both serine proteases. The proliferation of HT29 colon cancer cells was significantly affected by rTI1B in a dose-dependent manner (IC50 = 31 (sd 7) μm), whereas the inactive mutant did not show any significant effect on colon cancer cell growth. In addition, neither recombinant protein affected the growth of non-malignant colonic fibroblast CCD-18Co cells. These findings suggest that serine proteases should be considered as important targets in investigating the potential chemopreventive role of BBI during the early stages of colorectal carcinogenesis.