Objectives: The cyclophosphamide (CYP)-induced model of cystitis in mice closely fits the symptoms of chronic bladder inflammation. Cystitis was recently found to be due to an altered gap junction protein in a rat model. Thus, this study was conducted to evaluate changes in protein expression and composition in the bladder of CYP-treated mice. Materials and methods: Administration of CYP induced cystitis-related symptoms in mice. Cystometry was assessed and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured. Voiding interval values (time between voids) were assessed in mice under anesthesia. The bladders were removed for proteomic analysis using label-free quantitative proteomics and liquid chromatography–mass spectrometry. Additionally, immunochemistry (IHC) and Western blot were used to confirm the location and level, respectively, of ZO-2 expression. Results: Compared to the control group, the voiding interval values and urothelial thickness in the bladder in the CYP-treated group were significantly decreased. Additionally, we identified 105 differentially expressed proteins in the bladder of CYP-treated mice with proteomic analysis. These proteins were involved in cell–cell tight junctions, exocytosis, muscle development, contraction, and regulation, immune responses, proteolysis, and cell adhesion. IHC and Western blot confirmed the downregulation of the tight junction protein ZO-2 in the urothelium of bladder. Conclusions: Our results suggest that downregulation in tight junction protein ZO-2 and urothelium damage may have a role in cystitis-related OAB. These changes could be related to the molecular mechanism of cystitis-related OAB.