Chinese scorpion Buthus martensii Karsch(BmK) venom is a rich source of neurotoxins which bindto various ion channels with high affinity and specificityand thus widely used as compounds to modulate channelgating. An excitatory insect toxin, BmK IT, is not conservedwith a glutamate residue at the preceding position of thethird Cys residue, and is a toxin with a non-glutamateresidue at the relevant position in the excitatory scorpion β-toxin subfamily. In this study, the mutants of recombinantBmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT Cterminal(TKSYCDVQIN) truncated) were achieved bysite-directed mutagenesis. Biological activity of BmK ITand its mutants confirmed these residues or peptides playedkey roles in BmK IT. BmK IT (I25E) could increase thesensitivity of BmK IT, but BmK IT(E15G) could decreasethe sensitivity of BmK IT on Sf9 cells. BmK IT truncatedC-terminal hydrophobic amino acids could cross thespecies boundaries and was effective on mammalian C6cells. To date, several excitatory insect toxins have beenisolated and identified from the venom of Buthus martensiiKarsch. However, no functional data are available andtherefore its classification in the family of excitatory insecttoxins remains putative and is just based on its highsimilarity with the other toxins of this family. These resultsverified I25, E15 and C-terminal (TKSYCDVQIN) inBmK IT played key roles in the interaction of the BmK ITand its receptor- sodium channels on the surface of insectcells and laid a foundation for further structural andfunctional analysis of BmK IT.