Voltage-gated sodium (NaV) channels play a key role in the initiation and propagation of action potentials in electrically excitable cells. There are nine subtypes in humans denoted hNav1.1–hNav1.9. Several of these NaV channels play critical roles in the pathophysiology of pain. Gain-of-function mutations in SCN9A, which encodes the pore-forming a-subunit of hNav1.7, cause painful neuropathies such as primary erythromelalgia and paroxysmal extreme pain disorder, whereas nonsense mutations result in a congenital insensitivity to pain1,2. Thus, subtype-specific blockers of hNav1.7 are likely to be useful analgesics for treating a wide spectrum of pain disorders. β-TRTX-Ps1a (Ps1a) is a 3-disulfide, 34-residue peptide isolated from the venom of the Chilean tarantula Paraphysa scrofa. Ps1a was reported to inhibit various NaV subtypes3, but its activity against hNav1.7 has not been studied. We produced Ps1a and mutants thereof by overexpression in the periplasm of E. coli in order to facilitate correct disulfide-bond formation. The recombinant Ps1a inhibited hNav1.7 with an IC50 of 185 ± 28 nM. The solution structure of Ps1a determined using high-resolution 3D/4D NMR experiments acquired using non-uniform sampling revealed that the toxin adopts an inhibitor cystine knot (ICK) motif with an unique bioactive face comprised of both hydrophobic and charged residues. Site-directed mutagenesis revealed four hydrophobic residues that are critical for activity against both hNav1.5 and hNav1.7. In contrast, mutations of Phe6 and several charged residues caused specific loss of activity against either hNav1.5.or hNav1.7. Mapping of the Ps1a pharmacophores for activity against hNav1.5 and hNav1.7 should allow us to engineer Nav1.7-selective peptides that might be useful analgesics.