To investigate the intestinal flora situation, and the levels of inflammatory cytokines and T cell subsets, and the pregnancy outcomes of pregnant women with gestational diabetes mellitus (GDM). Methods: The clinical data of 196 pregnant women from January 2016 to January 2018 were analyzed retrospectively, which included 132 healthy pregnant women in control group and 64 pregnant women with GDM in research group. The number of intestinal flora, such as enterobacter, enterococcus, bifidobacterium, lactobacillus, bacteroides, and fusobacterium, the levels of inflammatory factors interleukin-2 (IL-2), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and the levels of T cell subsets CD3+, CD4+, and CD4+/CD8+ of the women in the two groups were detected. Logistic multivariate analysis was used to determine the risk factors of GDM during pregnancy. Spearman correlation coefficient was used to analyze the correlation between intestinal flora number, the levels of inflammatory factors and T cell subsets of the women with GDM and their pregnancy outcomes. Results: The levels of IL-2, CRP, and TNF-α, and the number of enterobacter, enterococcus, bacteroidetes and fusobacterium of the women in the research group were significant higher than those of the women in the control group, while the levels of CD3+, CD4+, CD4+/CD8+, and the number of bifidobacteria and lactobacillus of the women in the research group were significant lower than those of the women in the control group. The incidence of neonatal hypoglycemia, macrosomia, and cesarean section of the women in the research group were significant higher than those of the women in the control group (all P<0.05). The number of lactobacillus and bifidobacterium, and the levels of IL-2, CRP, TNF-α, CD3+, CD4+, and CD4+/CD8+ were the risk factors of GDM of pregnant women (P<0.05). The number of lactobacillus and bifidobacterium, and the levels of CD3+, CD4+ and CD4+/CD8+ of pregnant women with GDM were negatively correlated with their rates of caesarean section, macrosomia, and neonatal hypoglycemia (P<0.05), while the levels of IL-2, CRP, and TNF-αof pregnant women with GDM were positively correlated with their rates of macrosomia and caesarean section (P<0.05). Conclusion: The pregnant women with GDM are accompanied by the decreased intestinal probiotics number, the decreased immune function, and the increased inflammatory response, and the abnormal intestinal flora, immune function and inflammatory response of the women can lead to adverse pregnancy outcomes. Therefore, target interventions are of great value in reducing the occurrence of GDM and adverse pregnancy outcomes.