Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage gated sodium channels (VGSC), but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3 and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC₅₀ of 1.2 ± 0.2 nM while Kv2 and Kv4 channels were insensitive to 1 mM gambierol. Onset of block was similar from either side of the membrane and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1-Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier binding to the lipid exposed surface of the pore domain thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this novel binding site may explain why most ciguatoxins activate VGSCs, while others inhibit Kv channels. This novel Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level, but also for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.