Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory bladder disorder with limited therapeutic options. Grape seed extract (GSE) has potent antioxidant and anti-inflammatory properties; however, its therapeutic application is limited by poor bioavailability. This study aimed to develop grape seed extract-loaded niosomes (GSN) and evaluate their protective efficacy against cyclophosphamide (CYCL)-induced bladder injury model relevant to IC/BPS. Twenty-eight female rats were randomly allocated into four groups (n = 7): control, CYCL-treated, CYCL + GSE, and CYCL + GSN. CYCL was administered intraperitoneally at 75 mg/kg on days 1, 4, and 7, while GSE and GSN were given orally at 100 mg/kg for eight consecutive days. Bladder tissues were examined using histopathology, ultrastructural assessment, immunohistochemistry, oxidative stress biomarkers, gene expression analysis, and molecular docking. CYCL induced marked urothelial injury, inflammatory infiltration, fibrosis, oxidative stress, apoptosis, and disruption of barrier-related markers, accompanied by increased NF-κB, IL-6, caspase-3, MDA, and nitric oxide levels and reduced E-cadherin, SOD, GSH, Nrf2, and ZO2 expression. GSE partially ameliorated these alterations, whereas GSN produced stronger protective effects, restoring bladder histoarchitecture, improving ultrastructural integrity, reducing inflammatory and apoptotic immunoreactivity, and enhancing antioxidant defense markers. GSN also increased Nrf2 and ZO2 mRNA expression, suggesting an association with Nrf2-related antioxidant responses and improved urothelial barrier integrity. Molecular docking further indicated favorable interactions of GSE phytoconstituents with GSK3-β and the KEAP1-Nrf2 complex. Overall, GSN enhanced the therapeutic efficacy of GSE against CYCL-induced bladder injury, possibly through modulation of oxidative stress, inflammation, apoptosis, and barrier-associated pathways. Further protein-level studies are required to confirm direct Nrf2 pathway activation.