Abstract: Objective To establish a heat shock protein 90α(HSP90α) expression-inhibited cell line and study the effect of lowered HSP90α level on cell stress response. Methods The recombinant plasimid pSilencerHSP90 containing the 21nt small interfering RNA of human HSP90α was subcloned, purified and identified by DNA sequence analysis before introduced into mouse fibroblast cell line NIH-3T3 by electroporation. After G418 selection, the positive clones were identified by immunofluorescence and Western blotting. NIH-3T3 cells were subjected to hyperthermia at 44 ℃ for 40 min to simulate oxidative stress, and flow cytometry was performed to analyze the effect of low-level HSP90 on DNA damage under stress condition. Results Immunofluorescence and Westen blotting showed lowered HSP90 levels in the transfected cells. Compared with the control cells, cells subjected to hyperthermia displayed intensified DNA damage. Conclusion Low-level HSP90α causes the cells to be more vulnerable to oxidative stress condition, and HSP90 content can be associated with cell protection against such condition.