Amitriptyline, a tricyclic antidepressant, is commonly used to treat depression and neuropathic pain, but its mechanism is still unclear. We tested the effect of amitriptyline on 5-hydroxytryptamine 3 (5-HT3) receptor currents and studied its blocking mechanism because the clinical applications of amitriptyline overlapped with 5-HT3 receptor therapeutic potentials. Using a whole-cell voltage clamp method, we recorded the currents of the 5-HT3 receptor when 5-HT was applied alone or co-applied with amitriptyline in cultured NCB-20 neuroblastoma cells known to express 5-HT3 receptors. To elucidate the mechanism of amitriptyline, we simulated the 5-HT3 receptor currents using Berkeley Madonna？？ software and calculated the rate constants of the agonist binding and receptor transition steps. The 5-HT3 receptor currents were inhibited by amitriptyline in a concentration-dependent, voltage-independent manner, and a competitive mode. Amitriptyline accelerated the desensitization of the 5-HT3 receptor. When amitriptyline was applied before 5-HT treatment, the currents rose slowly until the end of 5-HT treatment. When amitriptyline was coapplied with 5-HT, currents rose and decayed rapidly. Peak current amplitudes were decreased in both applications. All macroscopic currents recorded in whole cell voltage clamping experiments were reproduced by simulation and the changes of rate constants by amitriptyline were correlated with macroscopic current recording data. These results suggest that amitriptyline blocks the 5-HT3 receptor by close and open state blocking mechanisms, in a competitive manner. We could expand an understanding of pharmacological mechanisms of amitriptyline related to the modulation of a 5-HT3 receptor, a potential target of neurologic and psychiatric diseases through this study.