Activators of soluble guanylyl cyclase (sGC) interact directly with its prosthetic heme group, enhancing the enzyme responsiveness in pathological conditions. This study aimed to evaluate the effects of the sGC activator BAY 58-2667 on voiding dysfunction, protein expressions of α1 and β1 sGC subunits and cGMP levels in the bladder tissues after cyclophosphamide (CYP) exposure. Female C57BL/6 mice (20-25 g) were injected with CYP (300 mg/kg, i.p) to induce cystitis. Mice were pretreated or not with BAY 58-2667 (1 mg/kg, gavage), given 1 h prior to CYP injection. The micturition patterns and in vitro bladder contractions were evaluated at 24 h. In freely-moving mice, CYP injection produced reduced the micturition volume and increased the number of urine spots. Cystometric recordings in CYP-injected mice revealed significant increases of basal pressure, voiding frequency and non-voiding contractions (NVCs), along with decreases in bladder capacity, intercontraction interval and compliance. BAY 58-2667 significantly prevented the micturition alterations observed in both freely-moving mice and cystometry, and normalized the reduced in vitro carbachol-induced contractions in CYP group. Reduced protein expressions of α1 and β1 sGC subunits, and of cGMP levels were observed in CYP group, all of which were prevented by BAY 58-2667. CYP exposure significantly increased the reactive-oxygen species (ROS) generation in both detrusor and urothelium, and this was normalized by BAY 58-2667. The increased MPO and COX-2 activities in the bladders of CYP group remained unchanged by BAY 58-2667. Activators of sGC may constitute a novel and promising therapeutic approach for management of interstitial cystitis.