Abstract: Significant methodological differences exist in the isolation and culture ofPDLSCs(PDLSCs) from rat and human. The isolation and culture of rat PDLSCs is restricted by theobstructions ofthe small donor molars, relativelimitedperiodontal ligament sources, and the difficulty of tooth extraction. However, the establishment of rat model of periodontitis is widely usedin the basic research, the isolation and culture of rat PDLSCs are of vital necessity for exploring the pathology of periodontitis. Furthermore, they are of additional importance for finding out whether the environmental changesimpair biological properties of PDLSCsto cause serious damage of periodontal tissues and the difficulties in regeneration.On the other hand, after the establishment of the methodology in the isolation and culture of rat PDLSCs, theoretical basis of stem cell-based periodontitis treatment and periodontal regeneration could be provided. Therefore, it is vitally significant to summarize the isolation and culture of rat PDLSCs that have promising applicable prospects.