Objective: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes, and short and long-term maternal and neonatal cardiovascular risks. Our laboratory has shown that elevated levels of endothelial extracellular vesicles (EVs) in pregnant women is predictive of adverse pregnancy outcomes in type 1 diabetes. However, this matter has not been explored in GDM. We therefore investigated the impact of GDM on maternal vascular health, focusing on the mitochondria. Design and method: We determined the mitochondrial protein expression altered by GDM in rat kidneys by Western blot analysis. We examined the levels of circulating endothelial EVs and EV mitochondrial DNA (mtDNA) from rat plasma samples via flow cytometry and quantitative real-time PCR, respectively. We assessed the effects of high glucose exposure for 48 hours on endothelial mitochondrial function in human umbilical vein endothelial cells (HUVECs). Results: In a rat model of diet-induced GDM (Pereira, 2015 J Physiol), we observed significant decreases in expression of oxidative phosphorylation Complexes I (p < 0.05) and II (p < 0.01) in kidneys. We also observed a three-fold increase in late gestational levels of circulating endothelial EVs in GDM rats (p < 0.01) as well as increases in levels of EV-associated mtDNA, COX2 (p < 0.05) and ND2 (p < 0.01), suggesting mitochondrial dysfunction. In cultured HUVECs exposed to high glucose for 48 hours, we observed a significant decrease in Complex II expression (p < 0.01) and five-fold increases in EV mtDNA levels. This effect persisted even after removing high glucose exposure for 24 hours. Conclusion: These data show maternal endothelial dysfunction in GDM, accompanied by mitochondrial dysfunction and increased endothelial EV release. These findings suggest that endothelial mitochondrial health may represent a novel approach to reduce risks associated with GDM.