Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.