Gestational diabetes mellitus (GDM) is a worldwide public health problem. MicroRNAs (miRNAs) have been reported to be associated with GDM progression. We intended to figure out the function of miR-342-3p in the insulin resistance (IR) and liver gluconeogenesis in GDM. GDM mouse models were established by intraperitoneal injection of streptozocin. The expression of miR-342-3p and regulatory factor X3 (Rfx3) in placenta and pancreatic tissues of GDM mice were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). GDM mice were treated with lentivirus-mediated antagomir-miR-342-3p for miR-342-3p downregulation. Enzyme-linked immunosorbent assay, hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and periodic acid-Schiff staining were conducted to detect the influence of miR-342-3p knockdown on the levels of blood glucose, insulin, biochemical indices as well as the apoptosis and pathological changes in placenta or pancreatic tissues of GDM mice. The binding between Rfx3 and miR-342-3p was validated by dual luciferase reporter assays. miR-342-3p was upregulated and Rfx3 was downregulated in placenta and pancreatic tissues of GDM mice. Moreover, miR-342-3p bound with Rfx3 3'-UTR and therefore downregulated the expression of Rfx3. miR-342-3p expression was negatively correlated to Rfx3 expression in placenta tissues of GDM mice. In addition, miR-342-3p depletion decreased the levels of blood glucose, insulin, biochemical indices as well as restrained the apoptosis and pathological changes in GDM mouse placenta and pancreatic tissues. Furthermore, Rfx3 silencing countervailed the alleviative influence of miR-342-3p downregulation on IR and liver gluconeogenesis in GDM mice. Collectively, downregulation of miRNA-342-3p inhibits IR and liver gluconeogenesis in GDM by upregulating Rfx3, which may provide novel insight for GDM treatment.