With a worldwide prevalence of 9-15%, gestational diabetes mellitus(GDM) has been recognized as the most common medical complication during pregnancy. Exosomes are extracellular vesicles that have potential to be used as biomarkers and therapeutic delivery tools for GDM. Exosomal content is often parent cell-specific thus providing insight or “fingerprint” of the intracellular environment of the originating cell. Furthermore, exosomes carry can communicate intercellular messages and also have the ability to modify target cells. We investigated the exosomal miRNA profile across gestation in women with normal glucose tolerance (NGT) and GDM and determined the signaling pathways associated with changes in their miRNA profile. Plasma was collected at three times during pregnancy (<16 weeks, 22-30 and 33-37 weeks of gestation) from NGT and GDM women. Exosomes were isolated from plasma samples that were obtained from two independent cohorts of patients, which were used for the discovery (cross-sectional study design with n=18 per group) and validation (longitudinal study design with n=16 and n=8 for NGT and GDM, respectively) phases of this study. Exosomes were characterized during gestation using a wide range of methods, including i) quantification of different populations of exosomes present in maternal circulation using nanocrystals coupled with specific antibodies (e.g. placental, endothelial and adipose tissue markers); ii) isolation of placental exosomes from maternal circulation by immunoaffinity capture using anti-PLAP-coated beads; and iii) optimisation of exosomal miRNA and protein analysis by next-generation sequencing and SWATH Mass Spectrometry, respectively. Using a small RNA library and linear mixed modelling analysis, statistically, significant differences in the expression of 279 (NGT) and 308 (GDM) miRNA were identified across gestation. In addition, 175 miRNA were miRNAs deferentially expressed in NGT and GDM. The plasma expression of miR-10a-5p, miR-16-2-3p, miR-92a-3p, miR-151b, and miR-1910-5p were significantly different between NGT and GDM women across gestation in the validation cohort. Using a quantitative, data-independent acquisition mass spectrometry approach, we identified a set of proteins in skeletal muscle tissues from women with GDM associated with glycolysis and gluconeogenesis which could be targeted by the candidate miRNAs within circulating exosomes. In conclusion, the miRNA content in maternal circulating exosomes differs across gestation in women with GDM compared to NGT women, suggesting that exosomes may be involved in maternal metabolic adaptation to pregnancy through the delivery of bioactive miRNAs.