A trypsin inhibitor from Cleome gynandra seeds (CGTI) was extracted and purified to apparent homogeneity following ammonium sulphate fractionation and Trypsin-Affigel-15 affinity chromatography with a 13.11-fold purification and 14.87 % yield. CGTI was very active against bovine trypsin; on SDS-PAGE, it gave an apparent molecular weight of 14.6 kDa. With a neutral sugar content of 1.5% and no free sulfhydryl groups, it was found to be a glycoprotein. Antitryptic activity of CGTI remained unchanged over a broad range of temperatures (0-80ºC) and pH (3-12) and is relatively stable in the presence of 8M urea, 1% SDS, 6 M Guanidine hydrochloride and PNGase F for 24 h at room temperature. The kinetic analysis demonstrated a non-competitive form of inhibition with a Ki value of 0.40 ± 0.08 nM against bovine pancreatic trypsin, indicating potent inhibitory activity. The trypsin inhibitory activity of CGTI was lost after modification of amino groups with acetic anhydride, suggesting that CGTA is a lysine-active site inhibitor.