OBJECTIVE Increased consumption of folic acid is prevalent, raising concerns about possible adverse effects. This prospective study aimed to explore the associations between the duration of folic acid supplementation and the risk of gestational diabetes mellitus (GDM) in Chinese women. METHODS A total of 326 pregnant women were prospectively included for detailed information on folic acid supplementation during pre-pregnancy and early pregnancy, lipid profiles at 16-18 weeks, and subsequent GDM diagnosis at 24-28 weeks. Associations among folic acid supplementation, lipid profiles, and risk of GDM were analyzed using linear and logistic regression models, adjusting for potential confounders. RESULTS The incidence of GDM in participants was 10.1%. We observed a U-shape relation between duration of folic acid supplementation and risk of GDM. Women who did not take folic acid and took folic acid for >90 days had a higher incidence of GDM compared to those who took folic acid for <=60 days. Moreover, lipid profiles were positively correlated with duration of folic acid supplementation and risk of GDM. After adjusting for demographic characters, energy and nutrients intakes and lipid profiles, the adjusted OR of GDM comparing taking folic acid for >90 days with taking folic acid for <=60 days was 3.45 (95% CI: 1.01, 11.8). CONCLUSIONS This prospective study indicate a positive association among prolonged folic acid supplementation, lipid profiles in the second trimester, and risk of GDM. Future studies are warranted to verify the causal relationship and underlying mechanisms.