Scorpion venoms contain a variety of peptides toxic to mammals, insects and crustaceans. Most of the scorpion toxins have been isolated from the venoms of scorpions in the family Buthidae, but little interest has been paid to non-Buthidae scorpions. In this study, we isolated a short-chain insecticidal toxin (LaIT1) from the venom of the scorpion Liocheles australasiae belonging to the Hemiscorpiidae family. This toxin showed insect toxicity against crickets at a dose of 1.0 microg/insect, but no toxicity was observed against mice even after injection of 1.0 microg of LaIT1 via the intracerebroventricular route, suggesting that the effect of the toxin is insect-selective. Edman sequencing and mass spectrometric analysis revealed that the toxin is composed of 36 amino acid residues and cross-linked by only two disulfide bridges. The pattern of the disulfide bridges was assigned by LC/MS analysis after enzymatic digestion. LaIT1 shows no sequence homology to any other known toxins, suggesting that this toxin represents a novel structural motif class.