Kv1.3 K+ channels play a central role in the regulation of T cell activation and Ca2+ signaling under physiological and pathophysiological conditions. Peptide toxins targeting Kv1.3 have a significant therapeutic potential in the treatment of autoimmune diseases; thus, the discovery of new toxins is highly motivated. Among Kv1.3-specific toxins, Vm24 (α-KTx 23.1) is a 36-residue Kv1.3-blocker peptide from the venom of the scorpion Vaejovis mexicanus smithi. Vm24 blocks Kv1.3 channel with high affinity (Kd = 2.9 pM) and displays excellent, over ~1500-fold KV1.3 selectivity against several ion channels (Varga et al, 2012; Gurrola et al, 2012). However, at high peptide concentrations (over 10 nM) it also blocks other ion channels including Kv1.1, Kv1.2 and KCa3.1. Based on the transcriptome analysis of the venom gland of V. mexicanus smithi a novel synthetic peptide, sVmKTx was generated, containing 36 amino acid residues. sVmKTx shows high sequence similarity to Vm24, but contains a Glu at position 32 as opposed to Lys32 in Vm24 (Csoti et al, 2022). sVmKTx displays reduced Kv1.3 affinity (Kd = 770 pM) but increased selectivity for Kv1.3 over hKv1.2 (~9,000-fold) as compared to Vm24, other channels tested in the panel (hKCa3.1, hKv1.1, hKv1.4, hKv1.5, rKv2.1, hKv11.1, hKCa1.1, hNav1.5) were practically insensitive to the toxin at 2.5 μM. Molecular dynamics simulations showed that introduction of a Glu instead of Lys at position 32 led to a decreased structural fluctuation of the N-terminal segment of sVmKTx, which may explain its increased selectivity for Kv1.3. sVmKTx at 100 nM concentration decreased the expression level of the Ca2+-dependent T cell activation marker, CD40 ligand. The high affinity block of Kv1.3 and increased selectivity over the natural peptide makes sVmKTx a potential candidate for Kv1.3 blockade-mediated treatment of autoimmune diseases.