Aim: The present study was carried out to assess the effect of bone marrow mesenchymal stem cells expanded by tissue culture on non-collagenous bone matrix protein (osteopontin and osteocalcin) expression in regenerating periodontal tissues utilizing immunohistochemical analysis in an experimental animal model. Materials and Methods: The rabbit bone marrow mesenchymal stem cells (BMMSCS) were harvested from 3month old New Zealand white rabbit in the Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo.This was done according to Charles H et al, 2004. Multilineage differentiation potential were tested. The cells were expanded and loaded on Collagen sponge. Periodontal defects were induced at the mesial surfaces in the premolar/molar area in 20 New Zealand white rabbits. Within every rabbit, periodontal defects were randomly covered with gingival flab only, serve as control group (Group 1), Collagen sponge (Group 2), collagen sponge seeded with the BMMSCs(Group 3),the assessment of the newly regenerating tissues was carried out by immunohistochemical analysis of expression of osteopontin (OP) and osteocalcin (OC) ( Non- collagenous bone matrix proteins. Results: Isolated cells showed stem/progenitor cell characteristics. Cell-loaded scaffold group showed higher expression of osteocalcin and osteopontin in the regenerated bone and periodontal ligament than the other groups. Conclusion: The expression of matrix molecules osteopontin and osteocalcin associated with regeneration of periodontal tissues is stronger following implantation of periodontal defects with collagen sponge seeded with BMMSCs compared to collagen sponge alone or surgical access flap.