The accumulation of a number of per- and polyfluoroalkyl substances (PFASs) in ovarian follicular fluid (FF) has been documented, raising serious questions about their impact on female fertility. Here, we tested the hypothesis that a mixture of PFASs acts in a paracrine manner on granulosa cells (GCs) as a metabolism-disrupting chemical. We selected perfluorooctane sulfonate (PFOS
22.4 ng/mL), perfluorooctanoic acid (PFOA
14.5 ng/mL), perfluorohexane sulfonate (PFHxS
21.3 ng/mL), perfluorodecanoic acid (PFDA
0.9 ng/mL), perfluoroheptane sulphonate (PFHpA
0.6 ng/mL), perfluoroundecanoic acid (PFUnDA
0.4 ng/mL), and perfluorononanoic acid (PFNA
2 ng/mL), which were the most commonly detected PFASs in FF of women undergoing assisted reproductive technology treatment. Exposure of mouse GCs to the PFAS mixture decreased the amount of active mitochondria and the mitochondrial membrane potential, which correlated with a reduction in ATP production and inhibition of oxidative phosphorylation (OXPHOS). At the same time, expression of the mitochondrial membrane-associated steroidogenic enzyme 3-beta-hydroxysteroid dehydrogenase (3βHSD) and production of the major steroids progesterone and estradiol were decreased. In addition, expression and activity of superoxide dismutase 1 (SOD1), an enzyme that neutralizes reactive oxygen species (ROS), were decreased while ROS levels and lipid peroxidation were increased without cell death, indicating that the PFAS mixture had subtoxic effects. Our results show that PFAS mixtures, at concentrations similar to those found in human FF led to GC dysfunction by impairing mitochondrial function and steroid secretions and therefore may have implications for reproductive health.