Objective:To construct and indentify glucocorticoid receptor β (GRβ) shRNA stably transfected human glioma cell lines and investigate the effect of GRβ on glioma cell cycle and proliferation. Methods:The specific siRNA was designed according to human GRβ sequence and transfected into human glioma cells by LipofectamineTM 2000 reagent. Knock down of GRβ by siRNA was measured by RT-PCR and Western blotting assays. The in vitro synthesized siRNA was inserted into shRNA sequence and directionally cloned into eukaryotic expression vector pGPH1/GFP/Neo. After transfection,the stable G0306 cell lines were selected by G418. Western blotting assays,MTS and flow cytometry were applied to detect the effect of GRβ interruption on glioma cell proliferation and cycle,respectively. Results:GRβ siRNA effectively inhibited the expression of GRβ. The inserted sequence of shRNA cloned into eukaryotic expression vector pGPH1/GFP/Neo was confirmed by DNA sequencing. The stable GRβ shRNA cell lines showed growth inhibition in G1 period,and the cell proliferation ability was significantly decreased. Conclusion:Stable GRβ knockdown glioma cell line (G0306) was constructed successfully and GRβ affected glioma cell proliferation,which laid a foundation for further study of the role and mechanism of GRβ in the central nervous system tumors.