Protease inhibitors, which are widely distributed in all types of life forms, are generally considered to be one of the most abundant proteins and a defense mechanism. A protease inhibitor from tartary buckwheat seeds (TBTI-Ⅱ ), with specific trypsin-inhibitory activity, was obtained by Resource Q anion-exchange chromatography and Superdex G 75 gel filtration. SDS-PAGE analysis indicated that the approximate molecular weight was 9.0 kD. Amino-acid analysis showed that the TBTI- Ⅱ was composed of 80 amino-acid residues with a high content of glutamate, aspartate and arginine. The inhibitor had high thermostability and retained 67.6% of its activity after heating at 100℃ for 10 min. The inhibition constant Ki was determined to be 1.01× 10-4 mol/L. It was demonstrated that the inhibitor was able to have an effect on the growth of cotton bollworm larva, after being fed with the artificial diets mixed with the target inhibitor. The present study indicates that the trypsin inhibitor from tartary buckwheat seeds could be a new potential anti-insect factor.