The periodontal ligament (PDL) comprises adult stem cells, which are responsible for periodontal tissue regeneration. In the present study, we investigated the specific profile of the stem cells in the human PDL. Microscopic analysis demonstrated that PDL cells showed a fibroblastic appearance, forming flat and loose aggregates. PDL cells expressed embryonic stem cell-associated antigens (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4, NANOG, SOX2, and REX1, and alkaline phosphatase activity), as well as conventional mesenchymal stem cell markers. When PDL cells were cultured in the presence of all-trans-retinoic acid, the numbers of SSEA-3+ and SSEA-4+ PDL cells were significantly decreased, while that of SSEA-1+ was increased. SSEA-4+ PDL cells showed a greater telomere length and growth rate. SSEA-4+ PDL cells exhibited the potential to generate specialized cells derived from three embryonic germ layers: mesodermal (adipocytes, osteoblasts, and chondrocytes), ectodermal (neurons), and endodermal (hepatocytes) lineages. Our findings demonstrated that SSEA-4, a major antigen to distinguish human embryonic stem cells, could also be used to identify multipotent stem cells in the PDL. Hence, SSEA-4+ human PDL cells appear to be a promising source of stem cells for regenerative medicine.