Objective · To investigate the differences of intestinal flora between gestational diabetes mellitus (GDM) pregnant women and healthy pregnant women. Methods · In this study, 74 pregnant women the Department of Obstetrics in Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Aug. 2017 to Jan. 2019 were recruited, including 51 GDM pregnant women (GDM group) and 23 healthy pregnant women (healthy group). High-throughput sequencing was performed on bacterial gene sequences the fresh feces of all pregnant womenusing Illumina MiSeq platform. Linear discriminant analysis (LDA) and LDA EffectSize (LEfSe) were used to detect the α diversity and abundance of intestinal flora of the two groups. Results · Compared with the healthy group, the intestinal flora richness of pregnant women in the GDM group was significantly higher (P0.027). Porphyromonadaceae and Alcaligenaceae in Family, and Bacteroides in Genus of microbiota in the GDM group were significantly higher (all P in Order, Lachnospirace in Family, and Neisseria, Streptococcus, Butyricicoccusm, Gardnerella and Atopobium in Genus of microbiota in the healthy group were significantly higher (all PConclusion · There are significant differences of intestinal flora between GDM pregnant women and healthy pregnant women.