<p>Early life stress leads to neurodevelopmental disorders. The excitatory-to-inhibitory functional switch of γ-aminobutyric acid (GABA switch) normally occurs on the up-regulation of K⁺-Cl^- cotransporter-2 (KCC2) in the first to the second postnatal week. GABA switch is necessary for normal construction of neural circuit. Therefore, early life stress may induce behavioral abnormalities through the influence of GABA switch. </p><p>　The purpose of this study is to examine the effect of maternal separation (MS) on the timing of GABA switch and on the adolescent behavior. </p><p>　Neonatal C57BL/6J male mice were divided into control group (Ctl) and experimental group. Experimental (MS) group was subjected to maternal separation for 3 hours a day during postnatal day 1 (P1) to P21. Then both groups were weaned at P22. We conducted quantification of KCC2 immunoreactivity (IR) at P7, 14, 21 and 35, and analyzed the timing of GABA switch by Ca²⁺ imaging method during P2 to P15. Behavioral analyzes such as open field, novel object recognition, elevated cross maze, attack action tests, were conducted from P35 to P38. MS resulted in a decrease in perimembrane KCC2-IR in the CA1 and CA3 regions at P7, 14, 21 and 35. NKCC1-IR in CA1 and CA3 were no significant difference between the Ctl and MS groups. Next, we analyzed the timing of GABA switch by Ca²⁺ imaging method. 10 μM of muscimol induced Ca²⁺ responses present in a higher percentage of neurons in the MS than in the Ctl on P8 and P11. The Ca²⁺ responses induced by muscimol were completely inhibited by pre-treatment with 10 μM of bicuculline. Compared to mice in the Ctl group on P35, MS group had decreased amounts of perimembrane KCC2 in hippocampal CA1 and CA3. There were no significant differences in NKCC1 in both groups on P35. We also found cognition and attention defects in MS group by behavioral analyzes. </p><p>　Taken together, neonatal MS inhibited the expression of perimembrane KCC2 for a long lasting period, leading to the delay timing of GABA switch in the hippocampus. The disrupted KCC2 expression could be associated with abnormal behaviors during adolescence.</p>