There is growing evidence suggesting that early life events have long-term effects on the neuroendocrine and behavioral developments of rodents. However, little is known about the involvement of early life events in the susceptibility to subsequent stress exposure during adulthood. The present study characterized the effect of maternal separation, an animal model of early life adversity, on the behavioral response to repeated restraint stress in adult rats and investigated the molecular mechanism underlying behavioral vulnerability to chronic stress induced by the maternal separation. Rat pups were separated from the dams for 180 min per day from postnatal day 2 through 14 (HMS180 rats). We found that, as young adults, HMS180 rats showed a greater hypothalamic-pituitary-adrenal axis response to acute restraint stress than nonseparated control rats. In addition, repeatedly restrained HMS180 rats showed increased depression-like behavior and an anhedonic response compared with nonrestrained HMS180 rats. Furthermore, HMS180 rats showed increased expression of REST4, a neuron-specific splicing variant of the transcriptional repressor REST (repressor element-1 silencing transcription factor), and a variety of REST target gene mRNAs and microRNAs in the medial prefrontal cortex (mPFC). Finally, REST4 overexpression in the mPFC of neonatal mice via polyethyleneimine-mediated gene transfer enhanced the expression of its target genes as well as behavioral vulnerability to repeated restraint stress. In contrast, REST4 overexpression in the mPFC of adult mice did not affect depression-like behaviors after repeated stress exposure. These results suggest that the activation of REST4-mediated gene regulation in the mPFC during postnatal development is involved in stress vulnerability.