1Department of Physiology and Membrane Biology, University of California Davis, Davis, CA, USA, 2Department of Pharmacology, University of California Davis, Davis, CA, USA, 3Department of Entomology and Nematology, University of California Davis, Davis, CA, USA, 4AnaBios, San Diego, CA, USA, 5AmbioPharm, North Augusta, GA, USA, 6Comprehensive Cancer Center, University of California Davis, Davis, CA, USA. The voltage-gated sodium NaV1. 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 NaV1. 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 NaV1. 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 hNaV1. 7 and more than 1,000-fold selectivity over human NaV1. 1, NaV1. 3, NaV1. 4, NaV1. 5, NaV1. 8, and NaV1. 9 channels. PTx2-3127 inhibits NaV1. 7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationallydesigned peptide inhibitors of human NaV1. 7 channels have transformative potential to define a new class of biologics to treat pain.