?-SIIIA, a novel ?-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of ?-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, ?-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin ?-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. ?-SIIIA is a potent, nearly irreversible blocker of Nav1.2 but also blocks Nav1.4 and Nav1.6 with submicromolar potency. The selectivity profile of ?-SIIIA, including poor activity against the cardiac sodium channel, Nav1.5, is similar to that of the closely related ?-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Nav1.2. The structural and functional characterization described in this paper of an analgesic ?-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.