Periodontal ligament fibroblasts (PDLFs) are important cells, which are involved in maintaining tooth integrity. Diabetes has been found to be associated with periodontal disease in a bidirectional manner. The aim of the present study was to investigate the stemness properties of human PDLFs (HPDLFs) in high glucose conditions. HPDLFs were analyzed for their osteogenic differentiation capacity by inducing the cells with osteogenic medium in various glucose concentrations. The gene expression was then examined using reverse transcription‑quantitative polymerase chain reaction analysis, and examinations of alkaline phosphatase activity and nodule formation were performed. The results of the gene expression analysis revealed that high glucose media induced the expression of NANOG, octamer-binding transcription factor 4, (sex determining region Y)‑box 2, cluster of differentiation 166 (CD166), PERIOSTIN and β‑CATENIN following culture of the cells for 3 days. Alkaline phosphatase activity increased following 14 days in the high glucose condition. In addition, higher numbers of calcified nodules were formed on day 28 in the group cultured with high glucose. The results showed that high glucose induced bone formation by elevating the expression of stem cell markers, particularly CD166, and this induction may be regulated through β-CATENIN.