The cotton bollworm, Helicoverpa armigera Hubner (Lep: Noctuidae), is an economically important pest of numerous major food crops worldwide. Protease inhibitors from plants, expressed constitutively in transgenic crops, have potential for pest management as an alternative to chemical pesticides. In this study, a protease inhibitor was isolated, purified, and characterized from Datura metel L. seeds. The purity of the isolated inhibitor was confirmed by reverse-phase high-performance liquid chromatography, and activity staining showed one major peak and one clear activity band for the protein. Electrophoretic studies following gel filtration and ion-exchange chromatography revealed two and one bands for purified proteins, respectively. Partial biochemical characterizations of the purified inhibitor were determined. Maximum inhibitory activity was observed at 40-45°C (optimal temperature) when tested against gut extracts of fourth to sixth instar H. armigera larvae. Thermo-stability of the trypsin inhibitor against sixth instar larval midgut trypsin was observed up to 50°C when incubated for 30 min and 2 h. Among metal ions tested, Fe2+, Cu2+, and Mn2+ were found to decrease the trypsin inhibitory activity, whereas Hg2+, Mg2+, K+, Zn2+, Na+, Ca2+, and Cd2+ were found to significantly increase the inhibitory effect. This trypsin inhibitor showed competitive inhibition where the apparent value of Michaelis-Menten Km increased, but the value of Vmax remained unchanged.