Maternal obesity is associated with complications of pregnancy and increases the infant's risk of developing obesity, diabetes and cardiovascular disease later in life. The placenta has an important role in determining the pregnancy outcome, and the syncytiotrophoblast (ST) is the main component of the placenta that supports the relationship between the mother and fetus. The differentiation of the cytotrophoblast (CT) into the ST is accompanied by changes in mitochondrial functions and dynamics. The objective of the present study was to investigate the effects of maternal obesity (without gestational diabetes) on the in vitro differentiation capacities of human CT isolated from term placenta by focusing on mitochondrial status. We found that, during human CT differentiation process, maternal obesity is associated with (i) a lower progesterone secretion, (ii) a transient impairment in the ST's fusion potential (via syncytin-2 and its receptor), (iii) a lower mitochondrial content, and (iv) weaker mRNA expression of oestrogen-related receptor-gamma (a key mitobiogenesis gene). Moreover, maternal obesity altered the time course of ATP and reactive oxygen species production throughout CT differentiation. The mitochondrial dysfunctions observed in isolated human CTs of obese women might explain the observed decrease in progesterone production. Our results demonstrated that obesity in pregnancy is associated with a functional impairment of the ST which might alter the foetal-maternal dialogue.