Objective To investigate the changes of serum miRNA-21 expression in patients with gestational diabetes and its mechanism on the biological behavior of trophoblast cells.Methods From January 2019 to October 2020, 66 pregnant women with diabetes in pregnancy and 38 healthy pregnant women with similar gestational weeks who underwent physical examination at the same time were selected as the study subjects. The expression level of miR-21 in serum of the two groups was tested by qRT-PCR. The JAR cell lines were transfected with miRNA to construct control, miR-21NC, miR-21, miR-21 inhibitor NC, and miR-21 inhibitor group cells. Cell viability was tested by cell proliferation detection kit 8 (CCK-8), cell proliferation was tested by Brdu, cell invasion was tested by Matrigel invasion test, and cell apoptosis was tested by TUNEL. The expression levels of Bcl-2, Bax, Cleared-capase 9, Vimentin, and E-cadherin were tested by Western blotting.Results The expression levels of FPG, miRNA-21, and HbA1c in the study group were higher than those in the control group (P < 0.05). The expression level of miR-21 in miR-21 group and miR-21 inhibitor group was higher and lower than that in control group (P < 0.05). CCK-8 test results showed that the cell viability of miR-21 group was lower than that of control group, and that of miR-21 inhibitor group was higher than that of control group and miR-21 group (P < 0.05). Compared with control group, the proliferation ability of cells in miR-21 group and miR-21 inhibitor group decreased and increased significantly (P < 0.05). The apoptosis rate of miR-21 group and miR-21 inhibitor group was significantly higher and lower than that of control group (P < 0.05). The number of penetrating cells in miR-21 group and miR-21 inhibitor group was significantly lower than that in control group (P < 0.05). Compared with the control group, the expression of E-cadherin and Vimentin in the miR-21 group was down and up, respectively, but the expression of E-cadherin and Vimentin in the miR-21 inhibitor group was down and up, respectively, compared with the control group and the miR-21 group (P < 0.05). The expression of anti-apoptotic protein Bcl-2 decreased in miRNA-21 group and increased in miR-21 inhibitor group (P < 0.05). Compared with the control group, the expression of Bax and Cleared caspased-9 protein in the miRNA-21 group increased, while the expression of the above proteins in the miR-21 inhibitor group decreased (P < 0.05).Conclusion There is high expression of miRNA-21 in pregnant women with gestational diabetes. miRNA-21 can promote cell apoptosis by regulating the inhibitory effect of Bcl-2/Caspase-9 on trophoblast cell proliferation and invasion.