Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa v1.2/β 1, rNa v1.4/β 1, hNa v1.5/β 1 and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK v1.1-1.2, hK v1.3, hK v11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na + influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K + efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK v1.3 was the most sensitive channel, with an IC 50 value 10.8±0.5 μM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.