Growing evidence suggests that metabolic abnormalities linked to maternal diabetes and obesity impair placental serotonin (5-HT) homeostasis. 5-HT is a multifunctional monoamine that plays an important role in placental and fetal development. Disturbances in placental 5-HT homeostasis alter placental structure and function and are associated with neurobehavioral abnormalities in the offspring. Here we investigated the effects of glucose and insulin, hallmarks of maternal diabetes and obesity, on the expression of key regulators of 5- HT homeostasis in the placenta, including the serotonin transporter (SERT), tryptophan hydroxylase 1 (TPH1), and monoamine oxidase A (MAOA). Studies were performed on the first trimester trophoblast cell line ACH-3P, at physiological oxygen levels characteristic of early placental development (2.5 and 6.5 %), and at ambient oxygen (21%). Expression of SERT and MAOA mRNAs was 25-45% lower and that of TPH1 was 15% higher at 6.5% compared with 2.5% oxygen. Furthermore, SERT and MAOA mRNA levels were increased by 2- to 3-fold and TPH1 mRNA levels by 20% at ambient (21%) compared to physiological (2.5% and 6.5%) oxygen levels. At physiological oxygen levels, treatment with glucose (25 mM) and insulin (10 nM) decreased SERT and MAOA mRNA expression by 10-35%. Conversely, at ambient oxygen, treatment with glucose increased SERT, TPH1, and MAOA mRNA levels 2- to 3-fold. The results suggest that oxygen changes in the first trimester of pregnancy, together with metabolic (glucose, insulin) alterations, may be a driving force for changes in placental 5-HT homeostasis. Oxygen is clearly a major determinant of placental 5-HT homeostasis to be considered in future in vitro studies.