Background: Guided tissue regeneration (GTR) is an accepted approach in the correction of periodontal bone loss. Nonetheless, the deficiencies of commonly applied absorbable membrane, such as flexibility and limited osteoconductive and osteoinductive capability, still leave much room for improvement. Thus, the feasibility of applying demineralized dentin tissue to improve the therapeutic effect of GTR in periodontal regeneration was explored. Methods: Demineralized dentin was harvested after acid treatment, and its physiochemical properties were assessed in terms of mineralization density, contact angle, three-point test, and cell attachment. Because of its similar characteristics with bone tissue, dentin that had been acid-treated for 6 hours was chosen to repair a periodontal defect using an induced-periodontitis canine model. Histologic measurements were taken to compare its therapeutic effects to an absorbable membrane group and an untreated group. Results: The demineralized dentin displayed continually decreased hardness and density as the acid etching time was prolonged. Enhanced attachment and spreading of bone marrow mesenchymal stem cells were observed on the 6-hour processed dentin. Furthermore, in the demineralized dentin group, more periodontal tissues were newly formed compared with the biomembrane and untreated groups. Conclusion: Acid etching represents an easy and promising approach to obtain demineralized dentin with desirable properties, similar to bone, for clinical application to promote periodontal tissue regeneration.