Abstract: OBJECTIVE To explore the association between imbalance of vaginal flora, human papillomavirus (HPV) infection and pathological grades of patients with cervical lesions and analyze their interaction effects. METHODS The women who received two-cancer screening in Nanjing Lishui People's Hospital from 2019 to 2022 were prospectively recruited as the research subjects, 320 of whom had cervical diseases were assigned as the cervical disease group, and 120 whose cervical cytology test were normal were assigned as the healthy control group. The association of imbalance of vaginal flora, HPV infection with pathological grades of cervical lesions was observed. RESULTS The incidence of HPV infection of the cervical disease group was 78.75%, higher than that of the healthy control group(P＜0.05); the proportions of patients with vaginal flora diversity and grade Ⅱ and Ⅲ density of the cervical disease group were 67.81% and 69.06%, respectively, lower than those of the healthy control group(P＜0.05); the proportion of patients with microecological abnormalities of the cervical disease group was 65.94%, higher than that of the healthy control group(P＜0.05). The higher the pathological grade of the cervical disease group, the poorer the status of flora was; from cervical intraepithelial neoplasia (CIN) Ⅰ to CINⅢ, the flora density and percentage of normal diversity declined(P＜0.05), the rate of microecological abnormalities was increased(P＜0.05). The higher the risk of HPV infection, the more severe the imbalance of flora was; the rate of microecological abnormalities of the high-risk HPV infection group was 84.80%, higher than 72.92% of the low-risk HPV infection group and the negative group (4.41%), and there was significant difference(P＜0.05). The result of Spearman correlation analysis showed that the HPV infection(r=0.652,P＜0.001), vaginal flora density(r=0.805,P＜0.001), virginal flora diversity(r=0.703,P＜0.001) and endovaginal microecologics(r=0.683,P＜0.001) were positively correlated with the cervical pathology. A 1-year follow-up for all of the patients showed that the lower the grade of cervical lesions, the better the prognosis was(Z=43.841,P＜0.001). CONCLUSIONS There is remarkable positive correlation between the imbalance of vaginal flora, HPV infection and the incidence of cervical lesions as well as the severity. There is interaction between the two, which jointly promote the progression of cervical lesions. The joint detection of vaginal microecology and HPV infection has great significance in assessment of risk of cervical lesions and prognosis.