Objective: Periodontitis affects nearly 90% of adults over the age of 70, resulting to periodontal tissue infection, destruction, and ultimately tooth loss. Guided tissue regeneration (GTR) is a method widely used to treat severe periodontal disease, and involves placement of an occlusive barrier to facilitate regeneration of the damaged area by periodontal ligament stem cells (PDLSCs). In this study, we evaluate natural extracellular matrix (ECM) as a scaffold material to provide a suitable microenvironment to support the proliferation, differentiation, and tissue-regenerating properties of PDLSCs.Design: The viability, proliferation, apoptosis, and migration of PDLSCs cultured on ECM membrane, that was isolated from porcine urinary bladders, were compared with those cultured on type I collagen membrane, a commonly used scaffold in GTR. To evaluate the effects of ECM vs. type I collagen on the tissue-regenerating properties of PDLSCs, the bio-attachment and cementoblastic/osteogenic differentiation of PDLSCs were evaluated.Results: Incubation of PDLSCs with ECM resulted in increased viability, proliferation, and reduced apoptosis, compared with type I collagen treated PDLSCs. Co-culture with ECM membrane also increased the migration and bio-attachment of PDLSCs. Incubation of PDLSCs with ECM membrane increased expression of the cementoblastic/osteogenic differentiation markers BSP, RUNX2, ALP, OPN, OCN, and periostin.Conclusion: ECM membrane enhances the proliferation and regenerative properties of PDLSCs, indicating that ECM membrane can serve as a suitable scaffold in the application of GTR to treat periodontal disease.