Purpose <p>This study aims to elucidate the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in Hunner-type Interstitial Cystitis (HIC) and evaluate its potential as a therapeutic target.</p> Methods <p>Bladder tissue samples were obtained from HIC patients and normal bladder tissue from bladder cancer patients. PACAP expression was assessed through immunohistochemistry. An in vitro HIC model was established using LPS-induced SV-HUC1 cells. PACAP knockdown was performed using siRNA. The expression of inflammatory markers (IL-6, IL-1β, TNF-α) and fibrotic markers (fibronectin 1, TGF-β1, collagen I) was evaluated via qPCR, Western blot, and ELISA. Cell migration and proliferation were analyzed using wound healing and CCK-8 assays. Transcriptomic profiling was conducted to identify differentially expressed genes (DEGs) and explore their functional significance.</p> Results <p>PACAP expression was significantly elevated in the bladder tissues of HIC patients. LPS stimulation of SV-HUC1 cells induced PACAP expression alongside increased levels of inflammatory cytokines, validating the inflammatory model. PACAP knockdown markedly suppressed IL-6, IL-1β, and TNF-α expression and attenuated LPS-induced fibrosis by reducing fibronectin 1, TGF-β1, and collagen I levels. Additionally, PACAP knockdown inhibited LPS-induced cell migration and proliferation, as evidenced by wound healing and CCK-8 assays. Transcriptomic analysis revealed distinct molecular alterations in HIC tissues, including PACAP upregulation, implicating it in HIC pathogenesis.</p> Conclusion <p>PACAP plays a pivotal role in the inflammatory and fibrotic pathways of HIC. PACAP knockdown alleviates LPS-induced pathological responses, highlighting its potential as a novel therapeutic target. Further research is warranted to investigate PACAP’s precise mechanisms in HIC and its translational application in clinical settings.</p>