Objective: To investigate the expression levels of miR-29, miR-372-3p, and miR-375 in gestational diabetes mellitus (GDM) and their correlation with insulin resistance (IR). Methods: A total of 220 GDM patients who were diagnosed and treated in our hospital from May 2019 to May 2020 were selected as a study group, and 220 pregnant women with normal glucose tolerance during the same period were selected as a control group. The fasting blood glucose (FBG), fasting insulin (FINS), and glycosylated hemoglobin (HbAlc) levels in pregnant women of the two groups were detected and compared, and the insulin resistance index (HOMA-IR) was calculated, the expression levels of miR-29, miR-372-3p and miR-375 in peripheral blood of pregnant women of the two groups were compared, and the correlation between them and IR was analyzed. Results: The level of FINS in the study group was significantly lower than that in the control group, and the levels of FBG, HbAlc and HOMA-IR were significantly higher than those in the control group (P<0.05); the expression level of miR-29 in peripheral blood of the study group was significantly lower than that of the control group, and the expression levels of miR-372-3p and miR-375 were significantly higher than those of the control group (P<0.05); the results of Pearson correlation analysis showed that the expression level of miR-29 in peripheral blood of the study group patients was negatively correlated with HOMA-IR (r=−0.379, P<0.05), and the expression levels of miR-372-3p and miR-375 in peripheral blood were positively correlated with HOMA-IR (r=0.530, 0.628, P<0.05). Conclusion: The expression levels of miR-29, miR-372-3p, and miR-375 in the peripheral blood of GDM patients are statistically different from those of pregnant women with normal glucose tolerance, clinical detection of their expression levels is of higher value for the diagnosis of GDM, and all of them are correlated to HOMA-IR of patients to some extent, and are involved in the occurrence and development of IR.