Background <p>The pivotal role of microRNAs (miRNAs) in the pathogenesis of gestational diabetes mellitus (GDM) have been increasingly highlighted in recent studies.</p> Aims <p>To investigate miR-144-3p expression in patients with GDM and its potential mechanisms in GDM.</p> Methods <p>According to the inclusion criteria, 98 pregnant women with GDM and 98 normal pregnant women were statistically selected. The levels of miR-144-3p were determined by RT-qPCR. The ROC curve was used to evaluate the diagnostic value of miR-144-3p for GDM. Pearson analysis was used to analyze the correlation. HRT-8/SVneo cells were induced by high glucose in vitro to mimic the high glucose environment of GDM. On this basis, the cell models of miR-144-3p overexpression and knockdown were established to investigate the potential mechanism of miR-144-3p in GDM.</p> Results <p>The level of miR-144-3p in GDM group was markedly higher than that in normal pregnant women. In terms of predicting GDM, miR-144-3p demonstrated high accuracy. MiR-144-3p was positively linked to blood glucose markers in GDM. Further investigation has confirmed that miR-144-3p harbors a binding site for FOXO1 and can directly modulate its expression. Moreover, miR-144-3p targeted FOXO1 to affect the proliferation of trophoblast cells.</p> Conclusion <p>The expression of miR-144-3p is elevated in pregnant women with GDM. MiR-144-3p may be involved in the regulation of GDM by targeting FOXO1.</p>