Objective To isolate,culture and identify a dog periodontal ligament stem cells(PDLSC) line in vitro. Methods The adult dog periodontal ligament cells were isolated by limited dilution of culture cell for single cell clone. Cells originated from one of these clones were assessed through colony-forming efficiency and immunocytochemistry assay and alkaline phosphatase stain was used to identify the source of adult dog periodontal stem cells, at the same time, PDLSC were induced with mineralizatin solution and was found to have long protrude like an osteoblast. Differentiation of PDLSC were assessed, Mineralized potential was studied by Von-Kossa staining. Results The dog PDLSC expressed STRO-1, which was the marker of mesenchymal stem cells. Also Vimentin, osteoblast-like marker alkaline phosphatase and Collagen-Ⅰ expressed weakly. Cells were clonegenic, highly proliferative cells and capable of differentiating into osteoblasts/cementoblasts. Conclusion The evidence suggests that the cultured cells were stem cells from adult dog periodontal ligament.