Amitriptyline, a tricyclic antidepressant (TCA) drug, is widely used in treatment of psychiatric disorders. However, the side effects of amitriptyline on vascular K+ channels remain to be determined. Therefore, we investigated the effect of the tricyclic antidepressant and serotonin reuptake inhibitor amitriptyline on voltage‐dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells, using the whole‐cell patch clamp technique. The Kv current amplitudes were inhibited by amitriptyline in a concentration‐dependent manner, with an apparent IC50 value of 2.2 ± 0.14 μM and a Hill coefficient of 0.87 ± 0.03. Amitriptyline shifted the activation curve to a more positive potential, but had no significant effect on the inactivation curve, suggesting that amitriptyline altered the voltage sensitivity of Kv channels. Pretreatment with Kv1.5 and Kv1.2 channel inhibitors did not alter the inhibitory effect of amitriptyline on Kv channels. Additionally, application of train pulses (1 and 2 Hz) did not affect amitriptyline‐induced inhibition of Kv currents, which suggested that the action of amitriptyline on Kv channels was not use (state)‐dependent. From these results, we concluded that amitriptyline inhibited the channels in a concentration‐dependent, but state‐independent manner. This article is protected by copyright. All rights reserved.