The voltage-gated sodium Na_V1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human Na_V1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human Na_V1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC₅₀s of 7 nM and 4 nM for hNa_V1.7 and more than 1000-fold selectivity over human Na_V1.1, Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.8, and Na_V1.9 channels. PTx2-3127 inhibits Na_V1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human Na_V1.7 channels have transformative potential to define a new class of biologics to treat pain.