Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. In the literature, phthalate exposure has been associated with preterm birth, low birth weight, and pregnancy loss, but information on possible mechanisms linking maternal phthalate exposure and placental development is limited. One hypothesis is the involvement of the peroxisome proliferator-activated receptor-γ (PPARγ), which belongs to the superfamily of nuclear receptors that regulate, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast (ST). The goal of this study was to investigate the action of mono-2-ethylhexylphthalate (MEHP) on PPARγ activity during in vitro differentiation of VCTs into ST, the essential tissue of the human placenta providing hormonal and exchange functions between maternal and fetal blood. Several techniques such as immunofluorescence, PPARγ activity / hCGβ assays, western blotting, and lipidomics analyses were combined to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10μM) on freshly isolated VCTs from human term placenta. 0.1 μM and 1 μM MEHP significantly decreased PPARγ activity and cell fusion index compared to controls, while, surprisingly, 10 μM had the opposite effect. MEHP exposure inhibited hCG secretion regardless of the concentration used and significantly altered lipid composition. This study suggests that MEHP: (i) disrupts trophoblast differentiation, (ii) alters the production of the essential pregnancy hormone (hCG), and (iii) that these effects are in part mediated by the nuclear receptor PPARγ. Thus, phthalates act as endocrine disruptors in the human placenta.