Voltage-gated sodium channel Nav1.7 is preferentially expressed in peripheral nociceptors. Clinical genetics, animal models and pharmacological studies indicate that Nav1.7 is an important target for analgesic drug development. In this paper, we report the isolation and characterization of APTX-1, a novel peptide toxin from the venom of spider Calommata signata Karsch. Patch-clamp analysis confirmed that APTX-1 potently inhibits the currents of Nav1.7. Mass spectrum results showed that the molecular weight of APTX-1 was 7 815.2 Da. The first 10 amino acid positions at the N terminal were ASCKQVGEEC. APTX-1 inhibited Nav1.7 currents in a concentration-dependent manner, with a half-inhibitory concentration of (0.46±0.08) μmol/L. Channel dynamics analysis showed that APTX-1 did not affect the reversal potential, voltage-dependent steady-state activation curve, and inactivation curve of Nav1.7, indicating that the peptide toxin did not affect the ion selectivity and voltage dependence of Nav1.7. In conclusion, we found a novel Nav1.7 peptide inhibitor, and the characteristics of its action on Nav1.7 were studied to provide lead molecules for the development of analgesic drugs targeting the Nav1.7.