Voltage-gated sodium channels (NaV) are molecular targets for the development of drugs for the treatment of diseases such as epilepsy, neuropathic pain, long QT syndrome, etc., as well as for insecticides. Therefore, the search for novel selective NaV channel ligands is relevant. Using amplicon deep sequencing of tentacle cDNA libraries from sea anemones Heteractis magnifica, 36 transcripts related to RpIII neurotoxin, a NaV channel modulators, were revealed. The recombinant RpIII was moderately toxic for mice (LD50 0.030 ± 0.004 mg/kg) but did not demonstrate any activity towards NaV in human SH-SY5Y cells. The toxin inhibited inactivation of heterologously expressed mammalian, insect, and arachnid NaV channels with higher specificity to insect channels. Cockroach (Blattella germanica) sodium channel BgNaV1 (EC50 of 2.4 ± 0.2 nM) and yellow fever mosquito (Aedes aegypti) channel AaNaV1 (EC50 of 1.5 ± 0.3 nM) were the most sensitive to RpIII, while mammals NaV had EC50 values above 100 nM except mNaV1.6 (EC50 of 43.8 ± 3.6 nM). The low nanomolar RpIII affinity to insect AaNaV1 may be explained by the extensive intermolecular contacts found by docking study. According to the predicted data, the toxin lands on the ion channel between voltage-sensing domain IV and pore domain I, also known as toxin site 3, followed by stabilizing the channels in the open state what was measured at electrophysiological experiments.