Objective To demonstrate the association between folate and gestational diabetes mellitus risk during the whole pregnancy. Design Retrospective cohort study. Setting A tertiary hospital. Population or Sample Pregnant women who received antenatal care. Methods A retrospective cohort study included 42478 women who received antenatal care in Shanghai, China from April 2013 to March 2017 was conducted. Serum folate and vitamin B12 were assayed before 24 gestational weeks and a diagnosis of gestational diabetes mellitus was made based on International Association of Diabetes and Pregnancy Study Groups criteria during 24-28 gestational weeks. General linear models were used to test the association between serum folate and plasma glucose and estimate the relative risks for gestational diabetes mellitus after controlling for different confounders. Restricted cubic spline was used to test the dosage-response relationship between serum folate and the risk of gestational diabetes mellitus. Main Outcome Measures Risks for gestational diabetes mellitus. Results Positive associations were observed between serum folate and plasma glucose levels consistently (fasting, 1-h, 2-h). The adjusted relative risks for gestational diabetes mellitus across serum folate quartiles were 1.00 (reference), 1.14 (95% CI: 1.04, 1.25), 1.41 (95% CI: 1.28, 1.54), 1.53 (95% CI: 1.40, 1.68), respectively (p for trend <0.001) regardless of vitamin B12 status and the gestational weeks when folate was measured. Conclusions The findings of the study are significant for improving the guidelines for folic acid supplementation during pregnancy from a gestational diabetes mellitus prevention perspective. Keywords Folate; Gestational diabetes mellitus; IADPSG; Restricted cubic spline; Cohort.