〔Abstract〕 Objective To analyze the relationship between vaginal microenvironment, high-risk human papillomavirus (HRHPV) infection and cervical intraepithelial neoplasia (CIN). Methods 80 CIN patients admitted to Zhaoqing Second People's Hospital from May 2018 to July 2020 were selected as the research object and setted as the observation group; in addition, 60 female healthy people with physical examination during the same period were selected as the control group. All subjects underwent vaginal microecological testing and real-time fluorescent polymerase chain reaction (PCR) testing. The pathogens, vaginal microenvironment indicators of the two groups of subjects were counted, as well as HR-HPV infection. Results The detection rate of aerobic vaginitis (AV), bacterial vaginosis (BV), and trichomonal vaginitis in the observation group were significantly higher than those in the control group, the vaginal cleanliness grade III ~ IV and the proportion of flora diversity grade I and IV in the observation group were also higher than those in the control group (P < 0.05); The positive rate of HR-HPV in the observation group was significantly higher than that of the control group, and the difference was statistically significant (P < 0.05). Conclusion Intravaginal pathogen infection, vaginal microenvironment changes and HR-HPV infection are important factors affecting the occurrence and development of CIN. Improving the vaginal microbial environment and strengthening HR-HPV screening have a positive effect on the prevention of cervical lesions.