microRNAs (miRNAs) have regulated the expression and function of genes implicated in many pathological settings, but their impact on the pathoetiological characteristics of overactive bladder (OAB) largely remains unknown. We have generated a mouse model in which adult mice can be induced for detrusor deletion of Dicer, an enzyme essential for miRNA processing. Targeted deletion of Dicer did not lead to a significant change for detrusor functionality under physiological conditions; however, loss of Dicer exacerbated cyclophosphamide-induced OAB, manifested by the higher severity of altered detrusor contractile force and sensitivity, abnormal urodynamics, and enhanced macrophage infiltration. Mechanistic studies revealed that loss of Dicer may impair the expression of miRNAs that are capable of targeting P2x mRNAs. As a result, mice deficient in Dicer manifest enhanced P2X expression in the detrusor on cyclophosphamide treatment, predisposing to the increased risk for OAB development. More important, studies using bladder biopsy samples of patients with OAB also demonstrated similar results as those found in animals. Taken together, our results suggest that miRNAs modulate OAB susceptibility by regulating purinergic signaling, in which the pathogenic insult induces the expression of miRNAs capable of targeting P2X mRNAs to suppress OAB symptoms. © 2012 American Society for Investigative Pathology.