Objective: To investigate the expression of Toll-like receptor 4 (TLR4) in placental tissues of pregnant women with gestational diabetes mellitus (GDM) and its relationship with placental histomorphological abnormalities and pregnancy outcomes. Methods: A total of 34 patients with gestational diabetes mellitus admitted to Rugao Hospital Affiliated to Nantong University were selected as a GDM group. A total of 40 normal pregnant women were selected as a control group during the same period. Hematoxylin eosin (HE) staining was used to detect the placental pathomorphology, and reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry was used to detect the expression of TLR4 in the placenta, and the relationship between the differences of each group and pregnancy outcomes was compared. Pearson single factor correlation analysis was used to analyze the correlation between TLR4 protein expression in placental tissues and placental pathological changes (P<0.05). Results: The incidences of fetal growth restriction and fetal distress in the GDM group were significantly higher than those in the control group. The incidences of poor villus maturation, arteriolar thickening of dry villi and capillary filling of placental villi in the GDM group were significantly higher than those in the control group. The level of mRNA expression in placenta TLR4 protein in the GDM group increased significantly, with a statistically significant difference (P<0.05) and the expression rate of TLR4 protein on maternal and fetal surfaces of the placenta in the GDM group was significantly higher than that in the control group (P<0.05). The expression of TLR4 protein was positively correlated with poor placental villus maturation, arteriolar thickening of dry villi, and capillary filling (P<0.05). Conclusion: The high expression of TLR4 protein in the placenta of GDM patients may related to the abnormality of placental histopathology and have adverse effects on pregnancy outcome.