α-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The α3β4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many α-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human β2 and β4 nAChR subunits that determine α-conotoxin selectivity for α3β4 nAChR. α-Conotoxin RegIIA was used to evaluate the impact of non-conserved human β2 and β4 residues on peptide affinity. Two mutations, α3β2[T59K] and α3β2[S113R], strongly enhanced RegIIA affinity compared with wild-type α3β2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in α3β4 had the contrary effect, emphasizing the importance of loop D residue 59 and loop E residue 113 as determinants for RegIIA affinity. Molecular dynamics simulation revealed the side chains of β4 Lys59 and β4 Arg113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the β2 Thr59 and β2 Ser113 side chains were not long enough to form such interactions. Residue β4 Arg113 has been identified for the first time as a crucial component facilitating antagonist binding. Another α-conotoxin, AuIB, exhibited low activity at human α3β2 and α3β4 nAChRs. Molecular dynamics simulation indicated the key interactions with the β subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual β subunit residues that critically determine affinity and pharmacological activity of α-conotoxins RegIIA and AuIB at human nAChRs.