A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K⁺ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K⁺ channel hK_V1.2 with high affinity (K_d = 65 nM). The conductance–voltage relationship of K_V1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin–channel interaction. Cm39 also inhibits the Ca²⁺-activated K_Ca2.2 and K_Ca3.1 channels, with K_d = 502 nM, and K_d = 58 nM, respectively. However, the peptide does not inhibit hK_V1.1, hK_V1.3, hK_V1.4, hK_V1.5, hK_V1.6, hK_V11.1, mK_Ca1.1 K⁺ channels or the hNa_V1.5 and hNa_V1.4 Na⁺ channels at 1 μM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.