Several micronutrients have been implicated in cervical carcinogenesis. However, their mode of action is still a matter of speculation. In particular, it is unclear whether certain nutrients reduce the probability of acquiring high-risk human papillomavirus (HPV) or whether they facilitate the clearance of high-risk HPV. We conducted a 24-month prospective follow-up study to test the hypothesis that systemic concentrations of folate are associated with the occurrence and duration of high-risk HPV infections after controlling for other micronutrients (vitamins B12, A, E, and C, total carotene) and known risk factors for high-risk HPV infections and cervical cancer. Circulating concentrations of these micronutrients and risk factors for cervical cancer were determined in a cohort of 345 women who were at risk of developing cervical intraepithelial neoplasia. Using the hybrid capture 2 (HC-2) assay, high-risk HPV status was evaluated at 6-month intervals up to 24 months. All women had at least three consecutive visit high-risk HPV test results. Higher folate status was inversely associated with becoming HC-2 test-positive [odds ratio (OR): 0.27; 95% confidence interval (CI), 0.08-0.91; P = 0.04]. Women with higher folate status were significantly less likely to be repeatedly HC-2 test-positive (OR: 0.33; 95% CI, 0.13-0.86; P = 0.02) and more likely to become test-negative during the study (OR: 2.50; 95% CI, 1.18-5.30; P = 0.02). To our knowledge, this is the first long-term prospective follow-up study reporting an independent protective role of higher folate status on several aspects of the natural history of high-risk HPV after controlling for known risk factors and other micronutrients. Improving folate status in subjects at risk of getting infected or already infected with high-risk HPV may have a beneficial impact in the prevention of cervical cancer.