We tested whether or not altered Ca2+ spark activity accounted for detrusor overactivity (DO) of Wistar rats after partial bladder outlet obstruction (PBOO). We constructed a DO model through PBOO and studied the Ca2+ spark activity of detrusor. By way of using confocal microscopy and the patch-clamp technique, Ca2+ sparks and spontaneous transient outward currents (STOCs) in detrusor myocytes were measured respectively. Our results indicated that Ca2+ spark activity and STOCs were significantly reduced in the DO detrusor myocytes compared to unafflicted control cells, and both of these had levels that were remarkably increased by applications of caffeine (10 μM), a RyR agonist, in DO myocytes. In addition, measures of detrusor contractions were also recorded by using freshly isolated detrusor strips. These results indicated that the spontaneous contraction of DO detrusor was significantly enhanced, and that the effect of caffeine (10 μM) upon detrusor contractions was reversed by applications of iberiotoxin (100 nM) which is a BK channel blocker. Western blotting (WB) analyses indicated that the levels of expression of ryanodine receptor type 2 (RyR2) and FK506 binding protein 12.6 (FKBP12.6) in bladder muscle were respectively decreased and increased in the samples from DO rats. Thus, we considered in the rat DO model wherein PBOO, the reduced Ca2+ spark activity in detrusor myocytes partly contributed to overactive detrusor contractions. The impaired Ca2+ spark activity may have resulted from decreased RyR2 expression and increased FKBP12.6 expression. Such novel findings in our research might help to provide means for better treatment outcomes for patients afflicted by bladder dysfunction.