The morbidity of tooth missing is the highest one among all the human organ diseases. The present restorations used in clinic, including fixed bridges, removable dentures and implant prosthetics, all exhibit their own defects, and hardly to restore the whole tooth structure and function. With the development of stem cells and tissue engineering, as an alternative, tooth regeneration, aiming at the generation of a structure like nature tooth, will be the therapeutic orientation to restore the lost tooth. The dental root, which supports the crown and occlusal force, is the fundamental part for tooth function. Based on the theory of tissue engineering, bio-roots were successfully generated by using mesenchymal stem cells (MSC) in miniature pigs. But the success rate of bio-root is not too high, is urgent to be improved for future clinic application. MSC mediated bio-root regeneration is depended on the dentinogenic differentiation regulation of MSC. Up to now, many factors affect the directed differentiation of MSC and further for the success rate of bio-root, including seeding cells, scaffold, growth factors and microenvironmental niche, etc. Microenvironmental niche is the key factor for affecting the MSC characteristics and special tissue regeneration. Basically, the bio-root is regenerated in jaw, while the jaw microenvironmental niche is prone to induce MSC for osteogenic differentiation, instead of dentinogenic differentiation. How to improve the dentinogenic differentiation of MSC in jaw microenvironmental niche is the key issue for increasing the success rate of bio-root.