To investigate the protective effect of dexmedetomidine (Dex) preconditioning against oxygen glucose deprivation/reoxygenation (OGD/R) injury in H9C2 cells and its regulation on intracellular calcium.　Methods　Rat cardiomyocyte cell line H9C2 cells were cultured until reached logarithmic growth phase. Cultured H9C2 cells were equally and randomly divided into five groups (n=6). Normal control group (group C), in which the cells were cultured as usual. Group OGD/R, in which the medium was replaced with the Dulbecco′s modified Eagle medium(without glucose, bubbled with 5% CO2 and 95% N2 for 30 min). An anoxia chamber was used to establish OGD/R injury. Cells were cultured in the anoxia chamber 24 h with 5% CO2 and 95% N2 to produce oxygen glucose deprivation and then followed by 3 h reoxygenation(returned to the aerobic environment) 3 h. Dex preconditioning groups (group D1, D2 and D3 with 0.1, 1.0 μmol/L and 10.0 μmol/L Dex respectively), Dex was performed before the process of OGD/R. After reoxygenation, the morphology of cells were observed with inverted microscope. The difference of cell viability was examined by 3-（4, 5-dimethyl-2-thiazolyl）-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) method, while the lactate dehydrogenase (LDH) release were detected by 2, 4-dinitrophenylhydrazine chromogenic method. The calcium imaging technology was used to detect intracellular calcium concentration. The mRNA level of FK506-binding protein 12.6 (FKBP12.6) was detected by the real-time quantitative polymerase chain reaction (RT-q-PCR).　Results　Compared with group C, the viability were of cells was markedly decreased, the LDH release and intracellular calcium concentration of cells were significantly increased in groups of OGD/R, D1, D2 and D3 (P<0.05), the mRNA level of FKBP12.6 was decreased in group OGD/R and group D2(P<0.05). Compared with group OGD/R, the cell viability was increased and the intracellular calcium concentration was decreased in group D2 and group D3(P<0.05), the LDH release was decreased and the mRNA level of FKBP12.6 was increased in group D2 (P<0.05).　Conclusions　 Dex preconditioning protects H9C2 cells against OGD/R injury, which may be associated with an inhibition of the intracellular calcium overload by up-regulation of FKBP12.6.