Periodontitis is one of the main complications of diabetes mellitus and many researches have been done on the relationship between periodontitis and diabetes mellitus, but too much are still unclear, especially the mechanisms by which high glucose induces damage of periodontal ligament fibroblasts. So in this study, we investigated the effects of different concentration of high glucose on apoptosis in human periodontal ligament fibroblasts and the possible mechanisms involved. Human periodontal ligament fibroblasts were cultured and subjected to glucose of different concentration (5.5, 15, 25, and 35 mM) for 24 h. Apoptosis was studied by flow cytometry, caspase assays, fluorescent real-time PCR and Western blot. We also determined Fas/FasL expression was by Western blot. The application of different concentration of high glucose induced a concentration-dependent increase of apoptosis and the activity of caspase-3 in cultured human periodontal ligament fibroblasts. Furthermore, inhibitor of caspase-3 could prevent the high-glucose-induced apoptosis in human periodontal ligament fibroblasts. Protein levels of Fas and FasL remained unchanged. These data indicate that high glucose induces a concentration- and caspase-3-dependent increase of apoptosis in cultured human periodontal ligament fibroblasts in vitro. Activation of caspase-3 caused by high glucose is independent of Fas/FasL signaling pathways system. These results suggest a novel mechanism for the regulation of human periodontal ligament fibroblasts apoptosis by high glucose.