Lipids, especially essential fatty acids (EFAs), play critical roles in guiding proper fetal development. Exposure to xenobiotics that may alter the fetal supply of EFAs/lipids could potentially lead to fetotoxicity. In this study, we investigated the effects of the peroxisome proliferator chemical, di-(2-ethylhexyl)-phthalate (DEHP), on the lipid metabolomic profile of the rat fetal brain upon maternal exposure during gestation. Female Sprague-Dawley rats were orally gavaged with a control vehicle or DEHP (1,500 mg/kg) from gestational day (GD) 0 to GD 19 and fetal brain tissue was isolated at GD 20. The concentrations of 11 lipid classes [free fatty acid, free cholesterol (FC), cholesterol ester (CE), diacylglycerol (DAG), triacylglyceride, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (PS), lysophosphatidylcholine (LYPC), cardiolipin, and sphingomyelin (SM)] were determined, as well as the differences in the composition of individual fatty acids. The total lipid concentration decreased with DEHP exposure, particularly for FC and SM, by 33 and 54%, respectively. The same trend was observed in the fatty acid compositions, particularly the unsaturated fatty acids, where a greater decrease was observed with longer fatty acid chain length. The compositions of docosahexaenoic acid decreased significantly in five lipid classes (P < 0.05), including CE (43%), DAG (60%), PS (33%), LYPC (35%), and SM (40%). In contrast, the most remarkable reduction of arachidonic acid presented in two lipid classes, CE and LYPC, with a decrease of up to 33%. These results suggest that in utero exposure to DEHP alters the lipid metabolome in the fetal brain, which may lead to aberrant neurodevelopment.