Mono(2-ethylhexyl) phthalate (MEHP), a bioactive metabolite of di(2-ethylhexyl) phthalate (DEHP), has been detected in the placenta and urine of pregnant women and is linked to adverse pregnancy outcomes. However, its effects on mitochondrial homeostasis in trophoblast cells remain incompletely understood. This study examined the impact of MEHP (0.5–200 µM) on mitochondrial function, dynamics, and biogenesis in human HTR-8/SVneo trophoblast cells. MEHP (≥5 µM) reduced MTT conversion without compromising membrane integrity, suggesting early metabolic or redox imbalance. A dose-dependent loss of mitochondrial membrane potential was observed, with increased reactive oxygen species (ROS) generation only at 200 µM. MEHP modulated the expression of mitochondrial dynamics genes, with a more pronounced mitofusin 1 (MFN1) induction at low doses and increased mitochondrial DNA content, suggesting a compensatory response to mild stress. Conversely, high doses more strongly induced fission and mitochondrial 1 (FIS1) expression, suggesting mitochondrial fragmentation. Both concentrations induced the expression of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and nuclear factor erythroid 2–related factor 2 (Nrf2), while sirtuin 1 (SIRT1) expression and activity declined progressively with dose. These results demonstrate that MEHP disrupts mitochondrial homeostasis in trophoblast cells at concentrations spanning the estimated human exposure range. The dose-dependent effects, from adaptive responses to overt dysfunction, may help explain the associations between MEHP exposure and placental pathology observed in epidemiological studies. © 2025 by the authors.