Jingzhaotoxin-34 (JZTX-34) is a selective inhibitor of tetrodotoxin-sensitive (TTX-S) sodiumchannels. In this study, we found that JZTX-34 selectively acted on Nav1.7 with little effect on othersodium channel subtypes including Nav1.5. If the DIIS3-S4 linker of Nav1.5 is substituted by thecorrespond linker of Nav1.7, the sensitivity of Nav1.5 to JZTX-34 extremely increases to 1.05 µ M.Meanwhile, a mutant D816R in the DIIS3-S4 linker of Nav1.7 decreases binding affinity of Nav1.7 toJZTX-34 about 32-fold. The reverse mutant R800D at the corresponding position in Nav1.5 greatlyincreased its binding affinity to JZTX-34. This implies that JZTX-34 binds to DIIS3-S4 linker of Nav1.7and the critical residue of Nav1.7 is D816. Unlike β -scorpion toxin trapping sodium channel in anopen state, activity of JZTX-34 requires the sodium channel to be in a resting state. JZTX-34 exhibitsan obvious analgesic effect in a rodent pain model. Especially, it shows a longer duration and ismore effective than morphine in hot pain models. In a formalin-induced pain model, JZTX-34 atdose of 2 mg/kg is equipotent with morphine (5 mg/kg) in the first phase and several-fold moreeffective than morphine in second phase. Taken together, our data indicate that JZTX-34 releases painby selectively binding to the domain II voltage sensor of Nav1.7 in a closed configuration.