The α3β2 and α3β4 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and peripheral nervous systems, playing critical roles in various physiological processes and in such pathologies as addiction to nicotine and other drugs of abuse. α-Conotoxin LvIA, which we previously isolated from Conus lividus, modestly discriminates α3β2 and α3β4 rat nAChRs exhibiting a ∼17-fold tighter binding to the former. Here, alanine scanning resulted in two more selective analogues [N9A]LvIA and [D11A]LvIA, the former having a >2000-fold higher selectivity for α3β2. The determined crystal structures of [N9A]LvIA and [D11A]LvIA bound to the acetylcholine-binding protein (AChBP) were followed by homologous modeling of the complexes with the α3β2 and α3β4 nAChRs and by receptor mutagenesis, which revealed Phe106, Ser108, Ser113, and Ser168 residues in the β2 subunit as essential for LvIA binding. These results may be useful for the design of novel compounds of therapeutic potential targeting α3β2 nAChRs.