Abstract: α3β4 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets in diseases such as addiction, cancer, and obesity. In this study, by replacing three amino acids of the α3 subunit with the corresponding positions of the rα6 subunit simultaneously, an α3K152E, E184D, Q195T subunit mutant was constructed by PCR-mediated site-directed mutagenesis and its cRNA was obtained by in vitro transcription. The cRNA of mutant subunits mixed in equal molar ratios with β4 subunits were microinjected into Xenopus oocytes. The pharmacological activity and function of α3K152E, E184D, Q195Tβ4 nAChR was evaluated by a two-electrode voltage clamp electrophysiological technique. Acetylcholine, nicotine, and cytisine were used as agonists to evaluate the magnitude of ligand-gated currents and gating characteristics of wild-type and mutant α3β4 nAChRs. The half-maximal effective concentrations (EC₅₀) of acetylcholine, nicotine, and cytisine on wild-type α3β4 nAChRs were 277.5, 34.02 and 23.05 µmol·L^-1, respectively, while their EC₅₀ values with α3K152E, E184D, Q195Tβ4 nAChR were 170.5, 26.6, and 98.45 µmol·L^-1, respectively. Thus these EC₅₀ values for the three agonists towards the mutant receptor were changed 0.6-fold, 0.8-fold, and 4.3-fold, respectively, compared with the wild-type receptor; cytisine was most strongly affected, with a 77% decrease in potency. However, the maximum agonistic efficiency (E_max) of cytisine on wild-type and mutant α3β4 nAChRs was increased from 94.12% to 155.08% relative to the peak current amplitude induced by 1 mmol·L^-1 acetylcholine. Thus, although the α3K152E, E184D, Q195Tβ4 nAChR had significantly reduced sensitivity to cytisine, the maximum current amplitude induced by cytisine was clearly increased. This mutant had slightly increased sensitivity to acetylcholine and nicotine. The results indicate that these three amino acids of the α3 subunit have important and varying effects on ligand binding to the α3β4 nAChR, providing a basis for further structure-functional research on α3β4 nAChR, as well as the pathology of related diseases.