Background <p>Vaginal intraepithelial neoplasia (VaIN) is a premalignant condition characterized by a high risk of persistence and recurrence, and no universally accepted standard treatment has been established. Photodynamic therapy (PDT) is a minimally invasive treatment option for VaIN, but its efficacy may be limited in some patients. We aimed to evaluate whether the addition of carbon dioxide (CO₂) laser ablation to PDT could improve the efficacy and safety of treatment for VaIN.</p> Methods <p>This multicenter retrospective comparative study included patients with VaIN treated at the First Affiliated Hospital of the University of Science and Technology of China and the First Affiliated Hospital of Bengbu Medical University between December 2018 and August 2024. Patients were assigned to the PDT plus CO₂ laser group or the PDT-alone group according to the treatment they actually received. All patients were followed for 12 months. The primary clinical outcome was complete remission (CR) at each follow-up endpoint. Secondary outcomes included HPV clearance among patients who were HPV-positive at baseline, cumulative recurrence, progression, and safety outcomes.</p> Results <p>A total of 146 patients were included, of whom 78 received PDT plus CO₂ laser and 68 received PDT alone. In the PDT-alone group, the CR rates at 3, 6, and 12 months were 70.6%, 63.2%, and 66.2%, respectively, compared with 70.5%, 85.9%, and 88.5% in the PDT plus CO₂ laser group. No significant between-group difference was observed at 3 months (<i>P =</i> 0.992), whereas the combination group showed significantly higher CR rates at 6 and 12 months (<i>P =</i> 0.002 and <i>P =</i> 0.001, respectively). Among patients who were HPV-positive at baseline, HPV clearance rates at 3, 6, and 12 months were 40.3%, 45.2%, and 50.0% in the PDT-alone group, compared with 67.6%, 66.2%, and 83.1% in the PDT plus CO₂ laser group, respectively. Cumulative recurrence by 12 months occurred in 6 patients (8.8%) in the PDT-alone group and 2 patients (2.6%) in the PDT plus CO₂ laser group.</p> Conclusions <p>In this multicenter retrospective comparative cohort study, CO₂ laser combined with PDT was associated with higher complete remission rates and greater HPV clearance than PDT alone in patients with VaIN. The combined approach may improve lesion control and was associated with a numerically lower cumulative recurrence rate within 12 months, although recurrence-related findings should be interpreted cautiously because of the small number of events. The treatment appeared generally tolerable based on available retrospective safety records. Further prospective studies are warranted to confirm these findings.</p> Trial registration <p>Not applicable. This study was a multicenter retrospective comparative cohort study and was not prospectively registered.</p>