Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65 Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1.