AbstractScorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαβ-motif. These toxins contain a toxin signature (K-C₄-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C₅-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks K_v1.2 and K_v1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C₅-R-N), is more potent in blocking K_v1.2 and K_v1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced K_v channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and K_v channel inhibitory function are not linked.