α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized α-conotoxins have a 4/7 loop size, and the major features of neuronal α-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel “molecular pruning” approach was undertaken to define the relationship between loop size, structure, and function of α-conotoxins. This involved the systematic truncation of the second loop in the α-conotoxin [A10L]PnIA [4/7], a potent antagonist of the α7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at α7 nAChRs, whereas 4/3[A10L]PnIA lost both α7 nAChR activity and helicity. In contrast, all truncated analogues lost ∼100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the α7 nAChR and AChBP.