Objective: To observe the effect of hypoxia on changes of proliferation ability of cultured human periodontal ligament fibroblasts (HPLFS) in vitro. Methods: HPLFS were randomly divided into two groups (experimental group and control group) by different oxygen concentrations. The oxygen concentration of control group was 21%. The oxygen concentrations of experiment group were 10%, 5% and 2%. The proliferation ability of HPLFS was detected by MTT colorimetric assay. The cell ultrastructure was observed by transmission electron microscope (TEM). Results: MTT assay results showed that compared with the control group, at the 12 h and 24 h, cell proliferation was enhanced with the hypoxia degree. At 24 h, cell proliferation showed significant differences. At 48 h and 72 h, proliferation of the cultured HPLFS in severe hypoxia group reduced significantly. Observed by TEM, at 24 h, not only the number of mitochondria, rough endoplasmic reticulum but also cell process increased in the cultured HPLFS in severe hypoxia group. At 72 h, the number of lysosome increased and the cell structure degenerated. Conclusion: Long-time severe hypoxia may lower the repair and remodeling abilities of periodontium, which might be one of the important etiological factors of periodontal disease under condition of high altitude.