Voltage-gated sodium (Na_V1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na_V1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na_V1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na_V1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na_V1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na_V1.7 channels, inhibiting Na_V1.7 with an IC₅₀ value of 0.3 nM, compared with IC₅₀ values of 30 to 150 nM for other heterologously expressed Na_V1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Aβ-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II (¹²⁵I-ProTx-II) binds with high affinity (K_d = 0.3 nM) to recombinant hNa_V1.7 channels. Binding of ¹²⁵I-ProTx-II is insensitive to the presence of other well characterized Na_V1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the ¹²⁵I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na_V1.7-selective blockers.