Introduction: The treatments of dentin hypersensitivity so far do not lead to satisfactory clinical outcome. Therefore, an ideal material has to be developed to treat dentin hypersensitivity effectively and efficiently. Calcium phosphate precipitation has exhibited potential value for the treatment of dentin hypersensitivity by occlusion of dentinal tubules. Objectives: The aim of this study is 1) to develop the gelatin-templated calcium silicate mesoporous (GCSM) biomaterial and evaluate its efficacy of dentinal tubules occlusion in vitro 2) to investigate its in vivo efficacy and biocompatibility. Materials and Methods: The GCSM biomaterial was synthesized using mesoporous silica as carrier, which was impregnated with a solution of CaO precursor and calcinated at 400°C for 5 hrs. We mixed GCSM with 30% H3PO4 and evaluated the efficacy of dentinal tubules occlusion by FE-SEM and XRD examinations. For in vivo efficacy and biocompatibility test, commercial product Seal & Protect, and negative control IRM were used to compare with GCSM. Results: GCSM/30% H3PO4 paste at pH 5.0-5.5 could occlude dentinal tubules by precipitates 30μm in depth without eroding dentin. XRD analysis revealed the main precipitate was CaHPO4(2θ=26.4°, 30.1°, 32.8°). As to the pulp irritation test, there is no significant difference among GCSM, Seal & Protect, and IRM (p>0.05). Conclusion: (1) pH=5.0-5.5 GCSM/30% H3PO4 paste could effectively occlude dentinal tubules without eroding dentin. (2) Animal study demonstrated that the developed GCSM paste could effectively occlude the dentinal tubules in vivo without irritating the pulp.