Purpose The aim of this study was to examine the association between periconceptional folic acid (FA) supplementation and risk of preterm birth. Methods We conducted a retrospective cohort study in women aged 18–49 who participated in the NFPHEP from 2010 to 2018, and had a singleton livebirth in 129 counties in southwest China. Participants were divided into four groups according to the time period starting FA use: no use, after the last menstrual period, at least 1–2 months before the last menstrual period, at least 3 months before the last menstrual period. The outcomes were preterm birth (gestation < 37 weeks) and early preterm birth (gestation < 34 weeks). Results 201,477 women were included and 191,809 (95.2%) had taken FA during periconception. Compared with women who did not take FA, women who started taking FA 1–2 months before their last menstrual period had a 15% lower risk of preterm birth (aOR = 0.85, 95% CI 0.79–0.92), and women who started taking FA at least 3 months before their last menstrual period had a 20% lower risk of preterm birth (aOR = 0.80, 95% CI 0.75–0.87), but women who started taking FA after their last menstrual period did not appear to reduce the risk of preterm birth. Conclusions In this study of 200,000 Chinese women, periconceptional supplementation with FA was associated with a lower risk of preterm birth. Women who started taking FA at least 3 months before their last menstrual period were more likely to reduce the risk of preterm birth.