It has been reported that Heteropodatoxin3 (HpTx3), a peptidic neurotoxin purified from the venom of the spider species Heteropoda venatoria, could inhibit Kv4.2 channels. Our present studynewly found that HpTx3 also has potent and selective inhibitory action on Nav1.7, with an IC50 of 135.61 ± 12.98 nM.Without effect on the current–voltage (I-V) relationship of Nav1.7, HpTx3 made minor alternation in the voltage-dependence of activation and steady-state inactivation of Nav1.7(4.15 mV and 7.29 mV, respectively) by interacting with the extracellular S3–S4 loop (S3b–S4 sequence) in domain II and the domain IV of the Nav channel subtype, showing the characteristics of both pore blocker and gate modifier toxin. During the interaction of HpTx3 with the S3b–S4 sequence of Nav1.7,the amino acid residue D in the sequence played a key role. When administered intraperitoneally or intramuscularly, HpTx3 displayed potent analgesic activity in a dose-dependent manner in different mouse pain models induced by formalin, acetic acid, complete Freund’s adjuvant, hot plate, or spared nerve injury, demonstrating that acute, inflammatory, and neuropathic pains were all effectively inhibited by the toxin. In most cases HpTx3 at doses of ≥ 1mg/kg could produce the analgesic effectcomparable to that of 1 mg/kg morphine. These results suggest that HpTx3 not only can be used as a molecular probe to investigate ion channel function and pain mechanism., but also has potential in the development of the drugs that treat the Nav1.7 channel-related pain.