Multipotent postnatal stem cells can be isolated from human periodontal ligaments (PDLs) and have the potential for large-scale expansion, offering a reliable cell source for clinical use in periodontal regenerative therapies. However, the effects of aging on the mesenchymal stem cell (MSC) properties of these cells remain undefined. The aims of this study were to isolate and characterize the periodontal ligament stem cells (PDLSCs) derived from human impacted third molars of donors of different ages and to compare their pluripotential capacity and regenerative potential. PDL tissues were obtained from 90 surgically extracted third molars and divided into four groups according to the donor's age. For each group, the colony-forming ability, proliferative capacity, migratory potential, cell surface antigens, differentiation ability, alkaline phosphatase activity, and gene expression of the PDLSCs were contrastively evaluated and quantified for statistical analysis. The in vivo tissue regenerative potential of PDLSCs was assessed by an in vivo ectopic transplantation model. It was found that human PDLSCs were successfully isolated and characterized as MSCs in all 90 teeth. PDLSCs derived from donors of different ages were successfully differentiated under an osteogenic and adipogenic microenvironment. The proliferative and migratory potential and the differentiation capacity of PDLSCs decreased as age increased (p < 0.05). PDLSCs derived from donors whose age is 62.6 ± 6.8 have a statistically significant decrease in pluripotential capacity compared with those derived from relatively young donors (p < 0.01). There is no identified cementum and PDL-like tissue formation in vivo among the two aging groups. We conclude that human PDLSCs could be successfully isolated from PDL tissue derived from donors of different ages, but the age-related changes of the MSC properties should be taken into account whenever they are intended for use in research or cytotherapy.