Background: During brain development, volatile anesthetic can rapidly interfere with physiologic patterns of dendritic development and synaptogenesis and impair the formation of precise neuronal circuits. KCC2 plays vital roles in spine development and synaptogenesis through its Cl- transport function and structural interactions with the spine cytoskeleton protein 4.1 N. The aim of this study was to dissect the mechanism of volatile anesthetics, which impair dendritic development and synaptogenesis via mediation of KCC2 cleavage. Methods: Westernblotting was employed to assess the expression change of NR2B, NR2A, calpain-1, calpain-2, KCC2, and 4.1 N protein of rat (PND 5). Co-immunoprecipitation was applied to demonstrate the interaction between KCC2 and 4.1 N protein. Long-term cognitive deficiency was assessed by MWM. Lentivirus-calpain-2 was administered by hippocampus stereotaxic injection. Results: There was a significant increase in the level of NR2B instead of NR2A exposure to isoflurane. Calpain-2 was excessively activated via NR2B after 6 h of isoflurane exposure. The expression of plasmalemmal KCC2 and 4.1 N protein was significantly decreased treated with isoflurane. The isoflurane group showed longer traveled distance, prolonged escape latency, less time spent in the target quadrant, and decreased platform crossings. Pretreatment with ifenprodil and downregulated calpain-2 expression significantly alleviated these neurotoxicity responses and cognitive deficiency after isoflurane exposure. Conclusions: A significant increase in NR2B, excessive activation of calpain-2 and increased cleavage of plasmalemmal KCC2, are involved in isoflurane-induced neurotoxicity and long-term cognitive deficiency. Blocking NR2B and calpain-2 activity significantly attenuated these responses. The KCC2 cleavage mediated by NR2B and calpain-2 is a major determinant of isoflurane-induced long-term cognitive deficiency.