Objective: To evaluate the histological modifications of the vaginal mucosa after repeated microablative fractional CO2 lasers treatments. As secondary objectives we evaluated the clinical effects associated with repeated microablative fractional CO2 lasers treatments using validated questionnaires. Design: Prospective intervention study. Setting: Division of Gynecology and Obstetrics, Urogynecology Unit, IRCCS San Raffaele Scientific Institute Sample: 15 postmenopausal women complaining of genitourinary syndrome of menopause symptoms. Methods: one cohort of patients submitted to at least two previous laser treatment cycles in the past years. Main outcome measures: Vaginal Health Index (VHI), Visual Analog Scale (VAS), Female Sexual Function Index (FSFI), Urinary Distress Inventory-6 (UDI-6), International Consultation on Incontinence Questionnaire – Urinary Incontinence (ICIQ-UI) and 5-point Likert scale. Moreover an histological examinations were carried out on all samples. Results: At 4 weeks after the last treatment VHI score and all FSFI items resulted significantly increased compared baseline. We observed a statistically significant decrease both in frequency and severity for all urinary symptoms after the follow up. We observed a statistically significant increase in the number of epithelial cell layer with a consequent increase in epithelial thickness, in the number of glycogen filled cells, and in the number of papillae, after the laser treatment. No signs of fibrosis were observed since neovascularization was observed in each single woman. Conclusions: This is the first study demonstrating the histological persistency of efficacy in repeated annually laser treatment cycles, with tissue changes always leading to regenerative results without any sign of fibrosis.