Abstract: Objective To investigate the protective effects of dexmedetomidine (Dex) against glutamate-induced cytotoxicity in PC12 cells and its mechanism. Methods PC12 cells were treated with varying concentrations of dexmedetomidine 1 h before exposure to a high concentration of glutamate. The cell viability was measured by MTT assay, and LDH release, MDA content and SOD activity were measured. The level of ROS was tested by DCFH-DA staining and flow cytometry. The level of intracellular Ca2+ was detected by Fluo-8 staining and flow cytometry, and the mitochondrial membrane potential (MMP) was determined with JC-1 staining and flow cytometry. Results Within the concentration range of 0.01 to 100 μmol/L, Dex dose-dependently protected PC12 cells against glutamate-induced cytotoxicity. Treatment with 100 μmol/L Dex significantly increased the cell viability to (86.6±2.2)% of that of the control cells (P<0.01) and decreased LDH release to 1.4±0.1 folds of the control level (P<0.01). In PC12 cells exposed to glutamate, Dex pretreatment significantly reduced MDA content (P<0.01), enhanced SOD activity (P<0.01), inhibited ROS overproduction (P<0.01), reduced intracellular Ca2 + level (P<0.01) and maintained a stable MMP (P<0.01). Conclusion Dexmedetomidine can protect PC12 cells against glutamate-induced injury possibly in relation with its anti-oxidative activity, inhibitory effect on intracellular calcium overload and protective effect of the mitochondria.