The increasing evidences showed that adverse early life events have profound long lasting consequences in adult rats including neural, behavioral, and cognitive effects. Early maternal separation was one of the models of adverse early life stress, but which period acts critically was unknown until now. The purpose of this paper was to explore the effects of maternal separation in different periods, that is, postnatal Day 2-9 and postnatal Day 14-21, on spatial learning and memory and long-term potentiation (LTP) in hippocampus of adolescent rats. Rat pups were assigned to three groups: early maternal separation from postnatal Day 2-9 (EMS2-9), separation from postnatal Day 14-21 (EMS14-21), and control (Con)--rats stayed with their mother all the time before weaning. Morris water maze test (MWM) and electrophysiological test were performed at 40-50 days of age. The results indicated that EMS14-21 impaired spatial learning and memory ability. For the excitatory postsynaptic potential long-term potentiation (EPSP LTP), both the two maternal separation groups showed decreased values compared to control group. In terms of population spike long-term potentiation (PS LTP), both the two maternal separation groups also showed lower values compared with control group, but only EMS14-21 group had significant difference compared with control group. In conclusion, our results revealed that EMS14-21 showed worst in both escape latency in Morris Water Maze test and LTP compared to control group and EMS2-9 group.