Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large-conductance Ca²⁺-activated K⁺ (K_Ca1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting K_Ca1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in K_Ca1.1 channel activity upon mAChR activation in freshly isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single K_Ca1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9 ± 1.5 years). Carbachol inhibited the amplitude and frequency of K_Ca1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca²⁺ from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a K_Ca1.1 channel inhibitor, indicating the critical role of the K_Ca1.1 channels. The potential direct carbachol effects on K_Ca1.1 channels were examined under conditions of removing the major cellular Ca²⁺ sources for K_Ca1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single K_Ca1.1 channel open probability and mean K_Ca1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state K_Ca1.1 currents. The data support the concept that mAChR activation triggers indirect functional K_Ca1.1 channel inhibition mediated by intracellular Ca²⁺, thus increasing the excitability in human DSM cells.