Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APETx3 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 42 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APETx3 is a natural occurring mutant from APETx1, only differing in 1 amino acid at position 3. APETx1 is believed to be a selective modulator of human ether-a-go-go related (hERG) potassium channels. In this study, APETx1, 2 and 3 have been subjected to an electrophysiological screening on a wide range of 21 ion channels expressed in Xenopus leavis oocytes: 10 cloned voltage-gated sodium channels (NaV1.2-NaV1.8, the insect channels DmNaV1, BgNaV1-1a and the arachnid channel VdNaV1) and 11 cloned voltage-gated potassium channels (KV2.1, KV3.1, KV4.2, KV4.3, KV7.1, KV7.2, KV7.3, KV7.4, KV7.5, hERG, the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels. However, the same substitution provides this toxin the ability to become a potent modulator of voltage-gated sodium channels (NaVs). APETx3 slows down the inactivation of mammalian and insect channels similar to site 3 toxins such as α-scorpion toxins and sea anemone NaVs toxins. Our screening reveals that the homologous toxins APETx1 and APETx2 display promiscuous properties as they are also capable of recognizing NaV channels, causing an inhibition of the sodium conductance.All together, these data provide new insights in key residues which allow these 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 believed to be highly selective.