Objective To analyze the changes and clinical significance of intestinal flora in patients with gestational diabetes mellitus (GDM). Methods A total of 100 GDM patients who underwent labor examination and delivery in Shiyan People′s Hospital of Bao′an District in Shenzhen City from September 2020 to June 2021 were selected as the GDM group. In addition, 80 normal pregnant women in the same period were selected as the normal pregnancy group to compare the intestinal flora of the two groups of pregnant women and adverse pregnancy outcomes of their mothers and infants. According to the maternal and infant pregnancy outcomes of GDM women, they were divided into the normal delivery group and the adverse pregnancy group to compare the intestinal flora detection results of the two groups. Results The colonies of Enterobacter and Enterococcus in GDM group were higher than those in normal pregnancy group,while the colonies of Bifidobacteria and Lactobacillus were lower than those in normal pregnancy group, the differences were statistically significant (P<0.05). The incidence of adverse pregnancy in GDM group was higher than that in normal pregnancy group, and the difference was statistically significant (P<0.05). The colonies of Enterobacter and Enterococcus in the adverse pregnancy group were higher than those in the normal delivery group, while the colonies of Bifidobacteria and Lactobacillus were lower than those in the normal delivery group, the differences were statistically significant (P<0.05). Conclusion Patients with GDM have intestinal flora disorder, and the incidence of maternal and infant adverse pregnancy outcomes is higher. Monitoring the changes of intestinal flora in patients with GDM has certain significance for clinical prevention and treatment of GDM and improvement of maternal and infant outcomes.