Early life stress is an important determinant for developing depression later in life. It is reported that maternal separation (MS) could trigger stress sensitivity in adulthood when exposed to stress again. However, it could also result in resilience to stress-induced depression. The conclusions are contradictory. To address this issue, C57BL/6N newborn pups were exposed to either daily short MS (MS for 15 min per day; MS15) or prolonged MS (MS for 180 min per day; MS180) from the first day postpartum (PD1) to PD21. Adult mice were then subjected to chronic unpredictable mild stress (CUMS) exposure from PD64 to PD105. The behavior tests such as the forced swimming test (FST), tail suspension test (TST), and open-field test were performed once a week during this time. Besides, the hippocampal neurosteroids, serum stress hormones, and hippocampal monoamine neurotransmitters were measured at PD106. We found that mice in the MS180 group displayed the reduced struggling time and the increased latency to immobility in both FST and TST. However, there was no significant difference in the MS15 group. The levels of hippocampal neurosteroids (progesterone and allopregnanolone) were decreased, and the serum levels of corticosterone, corticotropin-releasing hormone, and adrenocorticotropic hormone were overexpressed in the MS180 group. Besides, the expressions of monoamine neurotransmitters such as 5-hydroxytryptamine and 5-hydroxy indole acetic acid significantly decreased in the MS180 group, but not in the MS15 group. All findings revealed that prolonged MS, rather than short MS, could increase the susceptibility to depression-like behavior when reexposed to stress in adulthood. However, future studies are warranted to identify the underlying neuromolecular mechanism of the MS experience on the susceptibility to adult stress reexposure.