The highly directional maternal-to-fetal transfer of essential fatty acids (EFAs) across the placenta plays a critical role in guiding proper fetal development. Exposure to xenobiotics that may alter the fetal supply of EFAs/lipids could lead to fetal toxicity. Since the placenta is the first fetal arising organ that regulates fetal fatty acid homeostasis, the fatty acid/lipid composition in the placenta may serve as an indicator of fetal composition. In this study, we investigated the effects of the peroxisome proliferator chemical di-(2-ethylhexyl)-phthalate (DEHP), a widely used plasticizer and ubiquitous environmental contaminant, and its selective metabolites, mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (EHA) on the lipid metabolome in a rat HRP-1 trophoblast model. The concentrations of ten lipid classes (cholesterol esters, diacylglycerol, triacylglycerides, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysophosphatidylcholine, cardiolipin, and sphingomyelin) were determined, as well as the individual fatty acid compositions, especially the ω-3 and ω-6 family of EFAs. The level of each lipid class was significantly increased upon exposure to the agents, with MEHP and EHA generally showing higher increases than DEHP. The same trends were observed in comparing the fatty acid compositions. For example, the ω-3/ω-6 fatty acids ratio did not change, although the levels of ω-3 and ω-6 fatty acids were significantly elevated upon exposure. These results suggest that DEHP and its metabolites can alter lipid metabolome in a rat placental cell line, implying that these compounds may contribute to aberrant placental EFA/lipid homeostasis caused by peroxisome proliferation, and potentially result in abnormal fetal development.