The Ly-6/neurotoxin superfamily (Ly6SF) is a group of proteins containing the characteristic pattern with four to five disulfide bonds that are essential to build the unique three-dimensional structure so-called three-finger domain (TFD). The Ly6SF members consist of either secreted proteins or membrane-attached proteins containing a GPI-anchor sequence. The TFD was first identified in the alpha-bungarotoxin, a snake neurotoxin with potent antagonistic activity to nicotinic acetylcholine receptors (nAChR). Recently it has been reported that some endogenously expressed Ly6SF proteins (such as Lynx1, Lynx2 and SLURP-1) modulate nAChR function, either as positive allosteric modulators or as antagonists. Furthermore, these Ly6SF members have been implicated as balancers of neuronal plasticity in the adult nervous system. In this study, to explore novel endogenous nAChR modulators, we tried to identify TFD-containing proteins from the genome database and found that there are 31 candidate genes of this protein family common in human and mouse. The functional relationship analyses among these 31 proteins by phylogenic tree prediction showed that LyPD2, LyPD6 and PSCA were identified as possible novel nAChR modulators. To analyze biochemical protein-protein interaction between each Ly6SF protein and α4β2 nAChR, we performed co-immunoprecipitation assay. Along with Lynx1, an already reported α4β2 nAChR antagonist, LyPD2, LyPD6 and PSCA were found to be co-immunoprecipitated with α4β2 nAChRs. Furthermore, the histological localization analyses of these Ly6SF genes in the mouse brain by in-situ hybridization revealed some Ly6SF genes were specifically expressed in the pyramidal cell layer in the hippocampal CA1 region. These observations suggest that the Ly6SF genes extracted from the genome database are potentially involved in higher-order brain functions through nAChR regulation.