Introduction Preeclampsia (PE) affects 5–8% of all pregnancies worldwide and is a leading cause of maternal and fetal morbidity and mortality. It is manifested in 2nd trimester by new onset maternal hypertension, proteinuria, edema, and in severe cases, seizures and end-organ failure. PE is often associated with fetal complications such as intrauterine growth restriction (IUGR), stillbirth, birth hypoxia and impaired neurodevelopment. The physiopathological process of PE begins with inadequate trophoblast invasion early in pregnancy (1st trimester) and reduced uteroplacental perfusion that results in local oxidative stress and maternal systemic inflammatory response. There is growing evidence for the involvement of bioactive sphingolipids, angiogenic and pro-survival Sphingosine-1-phosphate (S1P) and pro-apoptotic Ceramide (Cer) and their synthesizing/metabolizing enzymes in trophoblast differentiation and invasion, uterine angiogenesis; all the cellular and physiological processes necessary to establish healthy placenta. We hypothesize that dysregulation of sphingolipid homeostasis (Cer-S1P rheostat) during early placentation process may have effect on downstream signaling and may contribute to the onset of the disease. Objectives Demonstrate differential expression and enzymatic activity of key sphingolipids’ enzymes (SPHK, NSMase) in in vitro and ex vivo model of placental hypoxia (JAR cells or villous explants +/− hypoxia) and in term placentae and term chorionic villous explants from PE and normotensive patients. Methods The human term placentae and chorionic villous tissue were obtained from PE and normotensive patients (Clinica Davila, Santiago, Chile). The hypoxia was induced in JAR cells (human choriocarcinoma) and villous explants by oxygen deprivation (1% or 8% O2) for 3 up to 24h. The normoxic conditions (21% O2) were used as a control. Protein and mRNA levels in human placental and chorionic tissues were determined by Western blot and real-time PCR, respectively. Neutral sphingomyelinase (NSMase) activity was measured on HPLC by using labelled exogenous substrate (6-NBD-sphingomyelin). Results Our results demonstrate that synthetic/metabolic pathways of S1P and Cer are altered in human term placentae and placental chorionic villous explants of preeclamptic patients as compared to normotensive subjects. Angiogenic Sphingosine kinase-1 (SPHK1) and Sphingosine-1-phosphate receptors (S1PR1, −3) mRNA and protein are down-regulated in PE placentae and villous explants, suggesting that they might be involved in challenged neo-angiogenesis observed in preeclampsia. Moreover, in vitro and ex vivo studies clearly show up-regulation of SPHK1 under low oxygen tension (1% O 2 ) in contrast to lack of SPHK1 stimulation under the conditions of placental physiological hypoxia (8% O 2 ). This may indicate that SPHK and its product, S1P might be an important angiogenic mediators during early placentation process, which is severely affected in PE. On the other hand, ceramide synthase 5 (CerS5) of de novo pathway is increased in PE placentae, while Neutral sphingomyelinase-2 (NSMase-2) protein expression and enzymatic activity is decreased in PE placental speicemen. These results suggest that pro-apoptotic Cer is augmented via de novo pathway in PE complicated pregnancies, while NSMase-2 derived ceramide may serve other functions. Conclusion We postulate that bioactive sphingolipids and their synthetic/metabolic pathways may contribute to pathogenesis of preeclampsia and may serve in the future as novel early biomarkers of this disease.