Background: HPV vaccines have only been used in China for 8 years, and routine HPV test is not recommended before HPV vaccination, there is still a need to figure out HPV effectiveness, and the impact of pre-vaccination HPV infection status on the protective effect of HPV vaccines in Chinese women. Methods: From June 2022 to June 2023, women aged 18-50 years without history of cervical or uterine excision were recruited from three medical institutions. Basic characteristics were compared between vaccinated and unvaccinated participants, and inverse probability treatment weighting (IPTW) was used for confounding factors. HPV infection rates and vaccine effectiveness (VE) were calculated, and sub-group analysis in vaccinated women were conducted to explore impact of pre-vaccination HPV infection status. Results: After adjusting for group differences, the VE was observed as 76.1% (95% CI: 58.7%-86.2%) against new HPV16/18 infections among 2,285 participants. Older age and having a master’s degree or higher were protective factors, while increased parity and using oral contraceptives alone were risk factors for HPV16/18 infection. Women with unknown pre-vaccination HPV status had significantly higher post-vaccination rates of hrHPV (RR 4.278, 95% CI: 2.537-7.215) infections compared to those HPV-negative pre-vaccination. However, no significant difference in new hrHPV infection rates was observed between pre-vaccination HPV-negative and HPV-positive women. Conclusion: In addition to HPV vaccination, age, parity, using oral contraceptives alone, and master’s degree or higher were independent influencing factors of HPV16/18 infection. Pre-vaccination HPV infection status did not directly affect the protective effect of the HPV vaccine against uninfected types.