Complete and predicable regeneration of complex periodontal structures, which include cementum, periodontal ligament (PDL), and alveolar bone, has been a great challenge for periodontal researchers. It is generally believed that human PDL from the root surface contains stem cells (r-PDLSCs), which can enhance cementum/PDL-like tissues regeneration in vivo. In this work, PDL was found to possess asymmetrically distributed stem cells observed by long-term bromodeoxyuridine (BrdU) labeling. Putative stem cells from human PDL on the alveolar bone surface (a-PDLSCs) were then isolated and characterized. It was shown that a-PDLSCs exhibited strong proliferation capability and expressed high percentages of mesenchymal stem cell markers. Comparatively, a-PDLSCs had higher multilineage differentiation potential than r-PDLSCs with regard to both osteogenic and adipogenic differentiation. Alkaline phosphatase activity and the expression of mineralization-related markers of a-PDLSCs were also higher than those of r-PDLSCs. In vivo, a-PDLSCs could regenerate bone/PDL-like structures and repair critical-size defects created in calvarial bone of NOD/SCID mice. Autologous PDLSC-mediated periodontal regeneration showed that a-PDLSCs could accomplish reconstruction of alveolar bone more perfectly than r-PDLSCs. Our data suggest that PDLSCs may have quite different characteristics depending on locations. a-PDLSCs may take a synergistic effect with r-PDLSCs in periodontal regeneration.