Cone snails (Conus spp.) are predatory marine gastropods found in tropical or sub-tropical regions. Despite their slow movement, they can immobilise prey or predators quickly by injecting a potent venom. The venom has been found to be a cocktail of pharmacologically active molecules, dominated by cysteine-rich peptides referred to as conotoxins. Although many conotoxins have been identified and fully characterised from Conus spp., there is evidence there could be thousands more awaiting discovery. Many of the uncharacterised conotoxins are present in low abundance and have multiple disulfide bonds making them difficult to study. Sigma-GVIIIA conotoxin is one of these challenging conotoxins, which is only found in the defensive venom of C. geographus. It is S-superfamily peptide containing ten cysteines with a VIII cysteine framework. It acts as a selective and competitive antagonist of serotonin type 3 (5HT3A) receptor. Here we have used two-dimensional NMR spectroscopy to solve the three-dimensional structure of the S-sigma-GVIIIA conotoxin isolated directly from the cone snail. This is the first structure determination of a peptide in the S-superfamily structure as well as determination of the VIII cysteine framework disulfide connectivity. This information will be valuable for producing the peptide using solid phase methods by allowing selective protection of cysteine residues. Being able to produce larger amounts of the peptide is likely to progress our understanding of this complex drug lead and provide insight into cone snail ecology.