Periodontal ligament (PDL) cells are the most important cells in the healing of wounds and the regeneration of periodontal tissues. The response of PDL cells regarding cellular activity to high glucose concentration levels could be the key in understanding the events associated with the dental care of brittle diabetes. We studied the effect of high glucose concentration levels on the cellular activity of PDL cells from five non-diabetic patients in vitro. PDL cells were cultured for 14 days in a normal glucose medium (1100mg/l of glucose) or in a high glucose medium (4500mg/l of glucose) and a 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for cellular viability was also performed. In order to evaluate the differentiation of PDL cells to osteoblast-like cells, mineralized nodule formation was induced with supplemented media containing 50microg/ml of ascorbic acid, 10mM of beta-glycerophosphate and 100nM of dexamethasone for 21 days. High glucose significantly inhibited the proliferation of PDL cells and reduced the optic density of the MTT assay. Concerning the mineralized nodule formation, the percentage of the calcified area to the total culture dish of PDL cells in high glucose level was lower than that in the normal glucose medium. In conclusion, high glucose inhibits the proliferation and differentiation of PDL cells. The data provide an explanation for the delayed periodontal regeneration and healing in diabetic patients.