Serotonin signaling plays an important role in regulating development and functions of the placenta. We hypothesized that metabolic disturbances associated with maternal obesity and/or gestational diabetes mellitus (GDM) affect placental serotonin homeostasis. Therefore, we examined the effects of high glucose (25 mM) and insulin (10 nM)—two hallmarks of maternal obesity and GDM—on mRNA expression of key regulators of serotonin homeostasis, including serotonin transporter (SERT), tryptophan hydroxylase 1 (TPH1), and monoamine oxidase A (MAOA), in the first-trimester trophoblast cell line ACH-3P, focusing on oxygen levels characteristic of early human placental development. Glucose downregulated expression ofSERTandMAOAindependently of oxygen level and upregulated expression ofTPH1at 6.5% oxygen but not at 2.5% oxygen. Compared to 6.5% oxygen, 2.5% oxygen upregulatedSERTand downregulatedTPH1expression, with no effect onMAOAexpression. Insulin upregulatedSERTonly at 2.5% oxygen but had no effect onTPH1andMAOAexpression. These results suggest that maternal metabolic alterations in early pregnancy may be a driving force for changes in placental serotonin homeostasis.