To develop a novel strategy for sealing and obturating dental root canals by tooth-like tissue regeneration, premolars with mature root apices were freshly collected, and root canals were prepared by following the clinical protocols in vitro. The teeth were immersed in supersaturated calcium and phosphate solution containing gallic acid and fluoride. At certain intervals, the dental roots were taken out, and their mineral precipitates were characterised by scanning electron microscopy, energy-dispersive spectroscopy mapping, X-ray diffraction and transmission electron microscopy. The cytocompatibility of the mineralizing products were evaluated with rabbit bone-marrow-derived mesenchymal stem cells in vitro. Results showed that the precipitates were mainly composed of fluoridated hydroxyapatite with ahexagonal prism morphology. Fluoridated hydroxyapatite initially nucleated and grew from the root canal dentine surface to the root canal centre. The fluoridated hydroxyapatite precipitate and root canal dentine intergraded together such that the interface became hardly distinguishable. The fluoridated hydroxyapatite precipitate grew into and obturated the dentinal tubules. In the root canal, the regenerated fluoridated hydroxyapatite densely packed and bundled together with a c-axis extension. After 7 days of mineralisation, the root canal was completely obturated, and the apical foramen was sealed. The mineralizing products had good biocompatibility with the cells, and the cells grew well on the mineralized surface. Biomimetic mineralisation strategy provides a novel means to regenerate tooth-like tissue to seal the root canal system permanently other than by passive synthetic material filling.