Background <p>Post-prostatectomy urinary incontinence (PPUI) is a common complication for patients with prostate cancer after surgery. MicroRNA-330-3p (miR-330-3p) is down-regulated in stress urinary incontinence patients. However, its clinical role and regulatory mechanism in PPUI remain unknown.</p> Objective <p>To assess the clinical significance of miR-330-3p in PPUI and to explore the potential mechanisms via matrix metalloproteinase 2 (MMP2) regulation.</p> Methods <p>This study enrolled 135 ageing prostate cancer patients (86 without PPUI, 49 with PPUI). Reverse transcription PCR (RT-qPCR) was utilized to measure the levels of miR-330-3p, while Receiver operating characteristic (ROC) analysis was conducted to evaluate the predictive significance of miR-330-3p for PPUI. The proliferative of human urethral fibroblasts (HUFs) was assessed by Cell Counting Kit-8 (CCK-8) assay, while inflammatory cytokines were quantified via enzyme-linked immunosorbent assay (ELISA) kits. Western blot assay was employed to examine the protein levels of extracellular matrix (ECM) remodeling-related markers. The miR-330-3p/MMP2 interaction was validated by dual-luciferase assay.</p> Result <p>miR-330-3p was significantly downregulated in PPUI patients, with low expression predicting PPUI. In HUFs, miR-330-3p overexpression inhibited IL-1β-induced hyperproliferation, inflammation, and ECM degradation. Overexpression of MMP2 counteracted the influence of miR-330-3p mimic on HUFs.</p> Conclusion <p>miR-330-3p is a potential biomarker for PPUI and regulates the function of urethral fibroblasts by targeting MMP2.</p>