Objectives- To assess the ectopic bone formation in canine masseter muscle following the implantation of the natural bovine bone minerals (NBM) loaded with canine mesenchymal stem cells (MSCs).Design- Experimental study.Animals- four mongrel dogs.Procedures- Tripotent MSCs isolated from the canine bone marrow were loaded onto the NBM sponges and allowed to adhere. The cell-loaded scaffolds were then implanted in parallel with cell-free control scaffolds in masseter muscles of four mongrel dogs. Eight weeks after, the animals were sacrificed and the ectopic bone formation in implantation site was studied using the sections prepared from the parts of the muscle containing the implants. Furthermore, the amount of bone formation in two studied groups was quantified using Image-Pro Plust software.Results- The implants from the both groups were appeared to be encapsulated by fibrous tissue in implantation site which included some trabecular bone containing osteocyte and osteoblast. There were no indications of inflammation and foreign body reaction, nor were there any indications of cartilage tissue formation.In contrast to control, in MSCs group, lamellar bone was observed in some area. More importantly, in cell loaded scaffolds more amount of bone was formed compared to that of control cell free scaffolds (P<0.05).Conclusion and Clinical Relevance- Taken together it seems that in vivo bone forming capacity of the NBM sponges would be improved by loading it with MSCs.