Background: Prenatal stress (PS) can induce depression in offspring, but the underlying mechanisms are still unknown. Objective: The aim of this work was to investigate the mechanism that underlies PS-induced depressive-like behavior in offspring. Methods: A prenatal restraint stress procedure was developed in which pregnant rats at GD14 to GD20 were placed head-first into a well-ventilated bottle three times each day and for 45 min each time. Depressive-like behavior in the male offspring was examined using the sucrose preference test (SPT) and the forced swim test (FST). The level of glutamate and the expression levels of GluN2A, p-CaMKII and myelin basic protein (MBP) in the hippocampus of PS-susceptible (PS-S) offspring were also evaluated. To clarify the mechanism by which PS leads to depression in offspring, the effects of excessive corticosterone were also investigated using an in vitro “injured neuronal” model. Results: The glutamate level in the hippocampus of PS-S male offspring was significantly elevated compared to controls. The expression levels of GluN2A and p-CaMKII were also altered. In addition, the optical density of MBP staining and the expression levels of MBP mRNA and MBP protein were decreased, demonstrating impaired myelinization in the hippocampus. Treatment of PS-S offspring with the GluN2A receptor antagonist NVP-AAM077 resulted in antidepressant-like effects in the FST, as well as rescue of the MBP and p-CaMKII abnormalities. Conclusions: These findings indicate that GluN2A is a promising target in the development of pharmacotherapies for PS-induced depression.