Background: We aimed to assess gut bacterial biomarkers during early pregnancy and subsequent risk of gestational diabetes mellitus (GDM) in Chinese pregnant women.Methods: Based on the Tongji-Shuangliu Birth Cohort study, we conducted a nested case-control study among 201 incident GDM cases and 201 matched controls individual matched on age, gestational week, and date of fecal sample collection. Fecal samples were collected during early pregnancy, and GDM was diagnosed at 24-28 weeks of pregnancy. Community DNA isolated from fecal samples and V3-V4 region of 16S rRNA gene amplicon libraries were sequenced. Results: In GDM cases versus controls, Rothia, Actinomyces, Bifidobacterium, Adlercreutzia, and Coriobacteriaceae, and Lachnospiraceae spp. were significantly reduced, while Enterobacteriaceae, Ruminococcaceae spp. and Veillonellaceae were over-represented. It was also found that the abundance of Staphylococcus relative to Clostridium, Roseburia and Coriobacteriaceae as reference microorganisms were positively correlated with fasting blood glucose, 1-h postprandial glucose, and 2-h postprandial glucose levels. Conditional Logistic regression showed that 4 microbial taxa during early pregnancy were associated with subsequent GDM risk, including Actinobacteria, Coriobacteriaceae, genus 26 of Coriobacteriaceae and an unknown specie of the Coprococcus.Conclusion: Gut microbiota during early pregnancy was associated with subsequent risk of GDM. Several beneficial and commensal gut microorganisms showed inverse relations with incident GDM, while opportunistic pathogenic members involving in GDM development, where they positively correlated with clinical measurements on OGTT. Our study provided promising biological markers that may be used in monitoring pregnant women’s health and developing therapeutic interventions on GDM at early stage of gestation.