Three defensin-like peptides (DLPs) were isolated from platypus venom and sequenced. One of these peptides, DLP-1, was synthesized chemically and its three-dimensional structure was determined using NMR spectroscopy. The main structural elements of this 42-residue peptide were an anti-parallel β-sheet comprising residues 15-18 and 37-40 and a small 310 helix spanning residues 10-12. The overall three-dimensional fold is similar to that of β-defensin-12, and similar to the sodium-channel neurotoxin ShI (Stichodactyla helianthusneurotoxin I). However, the side chains known to be functionally important in β-defensin-12 and ShI are not conserved in DLP-1, suggesting that it has a different biological function. Consistent with this contention, we showed that DLP-1 possesses no anti-microbial properties and has no observable activity on rat dorsal-root-ganglion sodium-channel currents.