Jingzhaotoxin-V (JZTX-V), a 29-residue polypeptide, is derived from the venom of the spider Chilobrachys jingzhao. Its cDNA determined by rapid amplification of 3' and 5'-cDNA ends encoded an 83-residue precursor with a pro-region of 16 residues. JZTX-V inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons with IC50 values of 27.6 and 30.2 nM, respectively. Moreover, the toxin exhibits high affinity to the resting closed states of the channels. JZTX-V also inhibits Kv4.2 potassium currents expressed in Xenpus Laevis oocytes (IC50=604.2 nM), but has no effects on outward delay-rectified potassium channels expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of sodium channels by shifting the activation curves to the depolarizing direction and the inactivation curves to the hyperpolarizing direction. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. The phospholipid membrane binding activity of JZTX-V is also verified using intrinsic tryptophan fluorescence analysis as well as acrylamide-quenching assays. Importantly, human multiple sodium channel subtypes are attractive targets for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.