α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting Cav2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB-α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than ten-fold and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the Trp59-β4 and Lys61-β4 residues of loop D, which formed a putative binding pocket. AuIB analogues with Phe9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively 'avoid' nAChR receptors while targeting receptors involved with nociception.