The isoform of voltage-gated potassium channels KV1.2 is of interest because mutations in its gene are associated with various diseases, such as ataxia and epilepsy. Selective ligands are needed to study the function of KV1.2 in health and disease. In our work, we obtained such a ligand based on the known scorpion peptide toxin, charybdotoxin (ChTx, α-KTx1.1) from the venom of Leiurus hebraeus, by introducing a single amino acid substitution M29I into its structure. ChTx_M29I peptide was produced in a bacterial expression system. Its pharmacological characterization was carried out in Xenopus laevis frog oocytes expressing a panel of human KV1 channels. We found that, compared to the parent toxin, ChTx_M29I peptide showed lower affinity for KV1.1, 1.3, and 1.6 channels, while its activity against KV1.2 increased manifold. We attribute this effect to the interaction of the peptide with a specific channel residue (V381 in KV1.2). If there is a relatively small residue at this position, then an advantageous contact is formed that increases the affinity. ChTx_M29I peptide studied by us presents one of the highest affinity (with a half-maximal inhibitory concentration IC50 ≈ 6 pM) and selectivity among KV1.2 ligands (affinity for other isoforms is lower by 680 times or more).