A novel biomimetic mineralization system was designed to induce a layer of hydroxyapatite on a demineralized dentin surface. This system was constructed as follows. A layer of 0.5% agarose gel containing 0.26M Na2HPO4 was used to cover acid-etched dentin slices, followed by a layer of agarose gel without phosphate ions. Then a neutral 0.13M CaCl2 solution was added onto the ion-free gel surface. The mineralization system (dentin-agarose gel containing phosphate ions-CaCl2 solution) was kept in a water bath at 37°C, and the gel and CaCl2 solution were replaced at various intervals. The results showed that the deposited hydroxyapatite crystals densely packed to each other, completely covered the dentin surface, and occluded the dentinal tubules after 10 days of biomimetic mineralization in vitro. Therefore, this method may provide the experimental basis for dentin remineralization and for a new method to treat dentin hypersensitivity and dental caries. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.