Partial bladder outlet obstruction (PBOO) usually induces overactive bladder (OAB) associated with detrusor overactivity (DO) which is related to the increased contractility of detrusor smooth muscle (DSM). The pharmacological activation of small-conductance Ca2+-activated K+ (SK) channels dramatically attenuates DSM contractility. However, the role of SK channels in the PBOO DSM is less clear. Here, we tested the hypothesis that PBOO is associated with decreased expression and function of SK channels in DSM and that the activation of SK channels is a potential target to regulate DO. Two weeks after surgically inducing PBOO in female guinea pigs, cystometry indicated that DO was achieved. Using this animal model, we conducted quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and isometric tension recordings. The qRT-PCR experiments indicated that PBOO DSM had reduced SK channel mRNA expression. Isometric tension recordings showed a decreased inhibitory effect of NS309 on spontaneous phasic and electrical field stimulation-induced contractions via the activation of SK channels in PBOO DSM. This study presents the novel finding that PBOO is associated with attenuated DSM SK channel expression and function, which results in increased DSM contractility and contributes to DO. Therefore, SK channels could be a therapeutic target to control OAB.