Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. Protein kinase C (PKC) modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the interactions between PKC and BK channels in DSM remains unknown. Here, we provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies on DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with phorbol 12-myristate 13-acetate (PMA) inhibited the spontaneous transient BK currents (TBKCs) in native freshly-isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode), upon inhibition of all major Ca²⁺ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated the intracellular Ca²⁺ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. The results supported the concept that PKC activation leads to a reduction in BK channel activity in DSM via a Ca²⁺-dependent mechanism, thus increasing DSM contractility.