Venomous animals have evolved to produce peptide toxins that modulate the activity of voltage-gated sodium (Nav) channels. These specific modulators are powerful probes to investigate the structural and functional features of Nav channels. Here, we isolated and characterized δ-theraphotoxin-Gr4b (Gr4b), a novel peptide toxin from the venom of the spider Grammostola rosea. Gr4b contains 37-amino acid residues with six cysteines forming three disulfide bonds. Moreover, Patch-clamp analysis confirmed that Gr4b markedly slows the fast inactivation of Nav1.9, and it also inhibits the currents of Nav1.3-Nav1.7, but it does not affect Nav1.8. Gr4b significantly shifts the steady-state activation and inactivation curves of Nav1.9 to the depolarization direction, and increases the window current, which is consistent with the change of the ramp current. Furthermore, analysis of Nav1.9/Nav1.8 chimeric channel revealed that Gr4b preferentially binds to the domain III voltage-sensor (DIII VSD) and has additional interactions with the DIV VSD. The alanine-screening analysis indicated that N1139 and L1143 in DIII s3-s4 linker participate in toxin binding. Taken together, our study reported a novel spider peptide toxin that slows the fast inactivation of Nav1.9 by binding to a new neurotoxin receptor site-DIII VSD. These findings provide insight into a functional role of Nav channel DIII VSD in fast inactivation.