Mitochondrial activity is critical for maintenance of correct glucose homeostasis and alteration in mitochondrial content or function may progressively lead to the development of insulin resistance. Evidence on the possible role of mitochondria in the pathogenesis of gestational diabetes mellitus (GDM) is conversely scanty and inconsistent. The aim was to evaluated mitochondrial DNA (mtDNA) content in peripheral blood of pregnant women with GDM. We selected 25 pregnant women affected by GDM and 50 controls with physiological pregnancies. A blood sample was collected at 32-36 weeks' gestation, stored and thawed simultaneously. The mtDNA content was determined utilizing a quantitative real-time polymerase chain reaction by the Taqman method, using a genomic control and a target gene. Results are expressed as copy number per nuclear DNA. The median (interquartile range) mtDNA content in GDM and controls was 122 (107-198) and 170 (129-196), respectively (p = 0.039). The mtDNA content was also correlated to GDM treatment, self-blood glucose monitoring and newborns' weight, but these analyses failed to document any statistically significant association. Attenuated mitochondrial function may play a role in the development of GDM. Further experiments are required to definitely clarify whether this defect represents a primary event in the pathogenesis of the disease.