Voltage-gated K+ (KV) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric KV channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of KV2.1 and electrically silent KV channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric KV2.1, KV2.2, and KV4.2 as well as the heterotetrameric KV2.1/6.3 and KV2.1/9.3 channels, was used to examine the role of these KV channels in DSM function. RT-PCR indicated mRNA expression of KV2.1, KV6.2–6.3, KV8.2, and KV9.1–9.3 subunits in isolated DSM cells. KV2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the KV current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for KV current. However, ScTx1 (100 nM) decreased the activation time-constant of the KV current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric KV2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric KV2.1/silent KV channels. Current-clamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that KV2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.