Abstract: Objective To test the proliferation effect of Hmgb3 on lung cancer cell line A549 after doxycyline-induced Hmgb3 shRNA lentivirus was constructed and packaged. Methods Hmgb3 and GFP shRNA sequences were designed and inserted into Tet-pLKO-puro plasmid to package lentivirus to infect A549 cells. Stable infected cells were obtained by puromycin screening. The doxycyline concentration was optimized to obtain the best induction effect. Western blot was used to test the silencing effect and MTT was for proliferation. Results Hmgb3 shRNA lentivirus could significantly reduce Hmgb3 expression in A549 cells and the efficiency of silencing was up to 73% (P<0.05). In the meantime, the proliferation rate of A549 cells in experimental group(46%)was much lower than that in control group(94%)(P<0.05). Conclusion Inducible Hmgb3 shRNA lentivirus was successfully constructed and silenced effectively target gene in A549 cells. Interfered Hmgb3 expression could inhibit the growth of A549 cells.