Background and Objective:  Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self‐renewal, multipotency, and stem cell marker expression.Material and Methods:  PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self‐renewal, a differentiation assay for multipotency, immunostaining for STRO‐1, and fluorescence‐activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO‐1) were performed.Results:  Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single‐cell colonies, and 30% of these colonies displayed positive staining for STRO‐1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO‐1.Conclusion:  The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self‐renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO‐1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.