Huwentoxin-Ⅴ (HWTX-Ⅴ) is an insecticidal peptide purified from the venom of spider Selnocosmia huwena. HWTX-Ⅴ contains 35 amino acid residues, including six cystein residues that form three pairs of disulfide bond. The positions of the disulfide bonds of HWTX-Ⅴ have been determined. To map the disulfide bonds, native HWTX-Ⅴ was multi-proteolytically digested. The resulting peptides were identified by matrix assisted laser desorption/ionization time of flight mass spectrometry, and it indicated the presence of one disulfide bond Cys9-Cys21. The partially reduced peptides by using Tris- (2-carboxyethyl)-phosphine at pH 3.0 over 12 minutes were purified by reverse phase high-performance liquid chromatography, and then one -free thiod and two-free thiod fractions were collected. The free thiods were carboxamidomethlate by iodoacetamide at the concentration of 0.5mol/L.The locations of disulfide bond Cys2-Cys16 and Cys15-Cys28 were proved by comparing N-terminal sequencing analysis these partially reduced and alkylated HWTX-Ⅴ with that of the intact peptide. Finally, the three disulfide linkage of HWTX-Ⅴ could be assigned as Cys2-Cys16,Cys9-Cys21 and Cys15-Cys28.