Objective: To analyze the different fabrication methods and surface structure of treated dentin matrix (TDM) and demineralized dentin matrix (DDM) and their diverse function on promoting the proliferation and osteogenic differential capability of human periodontal ligament cells (hPDLCs). This study provides a preliminary basis for the treatment of periodontal bone defects with bone substitutes from teeth. Methods: TDM was made from human dentin matrices and demineralized incompletely by soaking in different concentrations of ethylene diamine tetra-acetic while DDM was made of human dentin matrices and demineralized completely by soaking in a hydrochloric acid solution followed by observation via SEM. The liquid extracts of TDM and DDM were collected according to the protocol of the International Standardization Organization (ISO 10993). Then, hPDLCs were divided into the following three groups: the TDM group (liquid extracts of TDM), the DDM group (liquid extracts of DDM), the control group (a-modified eagle medium with 10% fetal bovine serum), hPDLCs were cultured with liquid extracts of TDM or DDM, or a-modified eagle medium with 10% FBS). hPDLC proliferation was detected by a Cell Counting Kit-8 (CCK-8). The alkaline phosphatase (ALP) expression and calcified nodules of hPDLCs were tested. Results : TDM obtained a preferable surface structure compared to DDM due to more sufficiently exposed dentinal tubules and looser fiber bundles of the intertubular and peritubular dentin. Both TDM and DDM promoted the proliferation of hPDLCs compared with the control group, and the proliferation of hPDLCs was significantly greater in the TDM group compared to the DDM group (F = 36.480, P < 0.05). The ALP activity of hPDLCs in the TDM group was higher than the DDM group. After a 14-day osteogenic induction, Alizarin red staining mineral nodes were observed in both groups; however, the TDM group displayed more calcified nodules than the DDM group. Conclusion The advantages of TDM including the surface structure, proliferation and osteogenic differentiation of hPDLCs, are more prominent than those of DDM, suggesting that TDM is a potential promising bone graft substitute in periodontal regeneration.