Objective To investigate the relationship between maternal supplementation with folic acid/ multiple micronutrients containing folic acid (MM-FA) and gestational diabetes mellitus (GDM) risk. Design Prospective cohort study. Setting Haidian Maternal and Child Health Hospital, Beijing, China. Population 3,458 pregnant women and 653 cases of GDM were approached between October 2017 and December 2020. Methods Sociodemographic characteristics, lifestyle data, and information on folic acid supplementation were obtained from a structured questionnaire. GDM was diagnosed according to IADPSG criteria (2010). After adjusting for confounding variables, associations between folic acid/MM-FA supplementation and GDM risk were estimated using binary logistic regression analysis. Main outcome measures Incident GDM. Results Taking MM-FA periconceptionally was associated with a higher GDM risk (aOR 1.33; 95% CI 1.05–1.69) compared to exclusive folic acid supplementation. And this association was observed exclusively in women with a pre-pregnancy BMI < 24kg/m2 (aOR 1.39; 95% CI 1.06–1.82). In separate analysis of pre-conception supplementation, women without folic acid supplementation before conception were more likely to develop GDM than those taking folic acid alone (aOR 1.40; 95% CI 1.01–1.96). Conclusions Maternal MM-FA supplementation may enhance the risk for GDM. These findings indicated that pregnant women should to be mindful of the risk of iron and other micronutrients over-supplementation when using folic acid supplements. It’s recommended that women take pure folic acid in preference and begin taking them from pre-conception in accordance with the recommended guidelines. Key words Folic acid; Multiple micronutrients; Gestational diabetes mellitus; Cohort