Objective To study the characteristics of intestinal microflora in patients with GDM, and to analyze the diversity and abundance of intestinal microflora. To explore the relationship between GDM and the changes of intestinal microflora. Methods: Mid–term pregnant women of 24–28 weeks of gestation in the outpatient department of the Third Affiliated Hospital Zhengzhou University from February 2017 to December 2017 for glucose tolerance test (OGTT) were enrolled in the study. 13 consecutive patients who met the diagnostic criteria for GDM were the experimental group (GH group), 11 consecutive normal pregnant women who matched with age and excluded pregnancy complications were used as a control group (NH group), and collect fecal specimens. High–throughput sequencing of V4 region of bacterial 16s rRNA gene was performed using the Illumina MiSeq sequencing platform, and the results were analyzed by bioinformatics. Using 16S rRNA high–throughput sequencing technology to analyze the difference of intestinal microflora between GDM patients and healthy pregnant women, the results showed that the abundance of intestinal microflora in GDM patients was lower than that in healthy pregnant women, and the distribution of microflora is different. In the GDM group, the intestinal microflora structure was significantly different（P＜0.05. At the level of genera, the abundance of Aggregatibacter, Campylobacter, Desulfovibrio, Gemmiger, Leptotrichia, Neisseria, Odoribacteria, Oxalobacter, Porhyromonas, WAL_1855D were lower than that of healthy control group (P < 0.05). Among them, the abundance of Desulfovibrio, Gemmiger and Odoribacter was significantly reduced in GDM patients. LEfSe analysis indicated that methanobacteria, Archaea and Vibrio played an important role in the healthy group, while Haemophilus and Pasteurella played an important role in the gestational diabetes mellitus group. Conclusions: The abundance of intestinal microbiota during mid–pregnancy was decreased in GDM patients compared with healthy pregnant women, and the distribution of microflora among individuals varied greatly.