The occlusion of dentinal tubules is considered to be an effective strategy to treat dentin hypersensitivity. This in vitro study introduced mesoporous silica nanoparticles (MSNs) for tubular occlusion to achieve deeper sealing. Further, MSNs with independently encapsulated calcium and phosphates (as calcium and phosphate sources)(Ca2+/PO4 3−@ MSNs) were introduced to achieve improved efficacy of tubular occlusion and remineralization. MSNs or Ca2+/PO4 3−@ MSNs were proportionally mixed with distilled water to make their respective desensitizing slurries, which were used to treat dentin surfaces. The efficacy of tubular occlusion was evaluated using scanning electron microscopy (SEM) images and an image analyzer, and compared with that achieved with Green-Or™—a commonly used densitizer. The results demonstrated that both MSNs and Ca2+/PO4 3−@ MSNs almost completely occluded dentinal tubules and formed a deeper seal which penetrated about 105 μm deep into the dentinal tubules. Significant differences in tubular occlusion were observed between Green-Or™ densitizer and MSNs or Ca2+/PO4 3−@ MSNs. The novel MSNs-based nanomaterials showed great potential as a treatment option for dentin hypersensitivity.