LaIT2, composed of 59 amino acid residues, is a peptide toxin isolated from the venom of the Yaeyama scorpion, Liocheles australasiae. LaIT2 is toxic to insects but not most mammals. The N- and C-domains of LaIT2 are known to possess antimicrobial and insecticidal activities, respectively. However, the molecular mechanisms are largely unknown because of the lack of a three-dimensional structure of LaIT2. Thus, we elucidated the solution NMR structure of LaIT2. LaIT2 adopts a β-KTx-like two-domain structure, in which the N- and C-terminal domains form a random coil and an α-β-β motif, respectively. Trifluoro ethanol and liposomes titration experiments showed that the unstructured N-domain of LaIT2 has the ability to form an α-helix. The N-terminal helix is amphiphilic, and one side of the helix is positively charged. Measurements of the antimicrobial and insecticidal activities of LaIT2 mutants suggested K15 in the N-domain was found to be responsible for the antimicrobial activities, whereas L53 and L54 in the C-domain were key residues involved in the insecticidal activity. Moreover, K21 in the N-domain is important for both activities. Therefore, two domains are suggested that they work together to show antimicrobial and insecticidal activity.