Objective To investigate the detailed procedure and efficacy of multiple targeting CO2 laser ablation (MTLA-CO2) for the treatment of vaginal intraepithelial neoplasia (VaIN) and to obtain a stably high cure rate and low complication incidence. Design Patients with VaIN were recruited and received MTLA. After follow-up, we aimed to find the risk factors affecting the efficacy. Setting Obstetrics & Gynecological outpatients’ department of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology. Population Fifty-seven cases of LSIL(VaIN1) and 160 cases of HSIL(VaIN2,3). Methods Patients with VaIN were recruited and received MTLA. The detailed clinical data were recorded and the risk factors were analyzed. Main Outcome Measures The pathological cure and HPV clearance. Results The cumulative pathological cure rate was 92.0% and 96.0%, and the cumulative HPV negative rate was 77.5% and 75.5%, for the HSIL(VaIN2,3) and LSIL(VaIN1) groups respectively. Larger areas of lesions (p =0.083) and history of hysterectomy (p =0.037) were independent risk factors for pathological persistence. Menopause (p =0.006) and immunosuppression (p =0.059) were independent risk factors for HR-HPV persistent infection. Condemn use (p =0.002) was a protective factor against HR-HPV infection. It was proposed 3~5 times of laser ablation for HSIL(VaIN2,3) and 2~3 times of laser ablation for LSIL(VaIN1). Conclusions The MTLA for VaIN was effective and well-tolerated. The schematization of MTLA was decided according to the characteristics of VaIN lesions, high-risk factors and surveillance. Funding The National Natural Science Foundation of China (81974410, 81572571, 81630060, 82002769, 81230038). Keywords Laser ablation, targeting, VaIN