α-Conotoxins preferentially antagonize muscle and neuronal nicotinic acetylcholine receptors (nAChRs). Native α-conotoxins have two disulfide links, CI-CIII and CII-CIV, and owing to the inherent properties of disulfide bonds, α-conotoxins have been systematically engineered to improve their chemical and biological properties. In this study, we explored the possibility of simplifying the disulfide framework of α-conotoxins Vc1.1, BuIA, ImI, and AuIB, by introducing [C2H,C8F] modification to the CI-CIII bond. We therefore explored the possibility of using hydrophobic packing of standard amino acid side chains to replace disulfide bonds as an alternative strategy to nonnatural amino acid cross-links. The impact of CI-CIII disulfide bond replacement on the conformation of the α-conotoxins was investigated using molecular dynamics (MD) simulations and nuclear magnetic resonance chemical shift index study. Two-electrode voltage clamp techniques and MD simulations were used to study the impact of disulfide bond deletion on the activities of the peptides at human neuronal nAChRs. All disulfide-deleted variants except ImI[C2H,C8F] had reduced potency for inhibiting nAChRs. Our results suggest that the CI-CIII disulfide bond is important to stabilize the secondary structure of α-conotoxins as well as their interaction with neuronal nAChR targets. Results from this study enrich our understanding of the function of the CI-CIII disulfide bond and are useful in guiding future structural engineering of the α-conotoxins.