Conotoxins are widely distributed and important for studying ligand-gated ion channels. TxIB, a conotoxin consisting of 16 amino acids derived fromConus textile, is a unique selective ligand that blocks rat α6/α3β2β3 nAChR (IC50= 28 nM) without affecting other rat subtypes. However, when the activity of TxIB against human nAChRs was examined, it was unexpectedly found that TxIB had a significant blocking effect on not only human α6/α3β2β3 nAChR but also human α6/α3β4 nAChR, with an IC50of 537 nM. To investigate the molecular mechanism of this species specificity and to establish a theoretical basis for drug development studies of TxIB and its analogs, different amino acid residues between human and rat α6/α3 and β4 nAChR subunits were identified. Each residue of the human species was then substituted with the corresponding residue of the rat species via PCR-directed mutagenesis. The potencies of TxIB towards the native α6/α3β4 nAChRs and their mutants were evaluated through electrophysiological experiments. The results showed that the IC50of TxIB against h[α6V32L, K61R/α3]β4L107V, V115Iwas 22.5 μM, a 42-fold decrease in potency compared to the native hα6/α3β4 nAChR. Val-32 and Lys-61 in the human α6/α3 subunit and Leu-107 and Val-115 in the human β4 subunit, together, were found to determine the species differences in the α6/α3β4 nAChR. These results also demonstrate that the effects of species differences between humans and rats should be fully considered when evaluating the efficacy of drug candidates targeting nAChRs in rodent models.