APETx3, a novel peptide isolated from the sea anemone Anthopleura elegantissima , is a naturally occurring mutant from APETx1, only differing by a Thr to Pro substitution at position 3. APETx1 is believed to be a selective modulator of human ether‐á‐go‐go related gene (hERG) potassium channels with a K d of 34 nM. In this study, APETx1, 2, and 3 have been subjected to an electrophysiological screening on a wide range of 24 ion channels expressed in Xenopus laevis oocytes: 10 cloned voltage‐gated sodium channels (Na V 1.2–Na V 1.8, the insect channels DmNa V 1, BgNa V 1–1a, and the arachnid channel VdNa V 1) and 14 cloned voltage‐gated potassium channels (K V 1.1–K V 1.6, K V 2.1, K V 3.1, K V 4.2, K V 4.3, K V 7.2, K V 7.4, hERG, and the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels and provides this toxin the ability to become a potent (EC 50 276 nM) modulator of voltage‐gated sodium channels (Na V s) because it slows down the inactivation of mammalian and insect Na V channels. Our study also shows that the homologous toxins APETx1 and APETx2 display promiscuous properties since they are also capable of recognizing Na V channels with IC 50 values of 31 nM and 114 nM, respectively, causing an inhibition of the sodium conductance without affecting the inactivation. Our results provide new insights in key residues that allow these sea anemone toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins thus far believed to be highly selective.—Peigneur, S., Béress, L., Möller, C., Marí, F., Forssmann, W.‐G., Tytgat, J. A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins. FASEB J. 26, 5141–5151 (2012). www.fasebj.org