LRRC4 is a novel brain relatively specific gene and a member of LRR superfamily, which displayed significant down-regulation in primary brain tumor biopsies and has the potential to suppress brain tumor growth. The establishnent of LRRC4 with doxycycline (Dox) induced Tet regulating system in U251 glioblastoma cell line was reported. Firstly, Tet-on regulating plasmid was transfected into U251 cells and screened by G418 to construct the single-stable U251 Tet-on cell line. Low background and high expression clone was acquired by testing luciferase activity. Successively, pTRE-2hyg/LRRC4 plasmid was transfected into the clone and screened by hygromycin. Two positive clones were received by RT-PCR and Northern blot analysis. The positive clones showed well dose-response and time-response in expression of LRRC4 with Dox inducement. Results show that establishment of LRRC4 with doxycycline induced Tet regulating system in U251 glioblatoma cell line is successfully, which provides an ideal experimental platform for understanding the mechanism of LRRC4 in glioma tumorigenesis and development.