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Gestational diabetes mellitus (GDM) is prevalent worldwide, leading to a high risk of significant morbidity for both the mother and offspring with complications. Increasing evidences suggest that gut microbiota plays a role in the pathogenesis of GDM. Lifestyle modification is the cornerstones of GDM treatment. However, a number of patients whose blood glucose is not controlled by lifestyle modification still require exogenous insulin to control blood glucose. No observational study is available about the relationship between the gut microbiota in GDM patients and lifestyle modifications. Thus, we investigated the differences in gut microbiota between GDM patients with successful glycemic control (GDM1) and failure of glycemic control (GDM2) by lifestyle modifications. We sequenced the V3-V4 regions of 16S ribosomal ribonucleic acid (rRNA) gene from stool samples of 52 singleton pregnant women with 24–28 weeks of gestation. Our results showed that Blautia, Eubacterium_hallii_group, and Faecalibacterium in the gut microbiota showed significant differences among the normoglycemic mother, GDM1, and GDM2 groups, respectively. The combined diagnostic performance of Blautia, Eubacterium_hallii_group, and Faecalibacterium in differentiating GDM2 from GDM was considered as the most reasonable identification indicator. Gut bacteria may participate in the pathological development of GDM2 through the peroxisome proliferator-activated receptor (PPAR) signaling pathway. These results indicated that Blautia, Eubacterium_hallii_group, and Faecalibacterium had important characteristic changes in the gut microbiota of women with GDM2.