17-α-ethinyl-estradiol (EE2) is the estrogen-like semi-synthetic hormone contained in most combined contraceptives and drugs for hormone replacement therapy. Due to its interference with estrogen pathways, EE2 is classified as an endocrine-disrupting chemical (EDC). Considering its widespread use, EE2 is an environmental contaminant in soil and water. EE2 can cross both the placental and blood-brain barriers (BBB), reaching the developing hippocampus, a glutamatergic area that expresses hormone receptors. Hormones (e.g., estrogens, thyroid hormones) influence synaptogenesis and synapse development. This process is timely regulated by the N-methyl-D-aspartate (NMDA) receptor subtype 2B (GluN2B) to NMDA receptor subtype 2A (GluN2A) switch in glutamatergic synapses. This study investigates the EE2 effect on the expression and distribution of glutamatergic receptor subunits in rat primary hippocampal neurons exposed during their development, and the consequences on spine maturation and postsynaptic calcium transients. Neurons treated with EE2 following different exposure schemes are analyzed for NMDA receptor and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the homogenate and at the postsynaptic site. Results obtained suggest that EE2 alters the developmental program of the glutamatergic system when administered at 7 days in vitro (DIV). Depending on the exposure time window, altered expression (7 DIV 24h) or translocation to the postsynaptic sites (7–18 DIV) of selected NMDA and AMPA receptor subunits occurred. Moreover, morphological analyses by confocal microscopy in transfected neurons revealed a decrease in mature mushroom-shaped and an increase in thin-shaped spines, while bicuculline, a GABAA blocker, induced an enhancement of postsynaptic calcium transients in control but not in EE2-treated neurons (7–18 DIV).