The aim of this study is to evaluate intratubular crystal formation from the experimental material consisting of dicalcium silicate (C2S) and tricalcium silicate (C3S) with nano-scaled particle size. A total of twenty-four specimens were made by isolating 8 mm of the cervical part centered at the cementoenamel junction of extracted premolars. Twelve specimens were not treated and considered as control. The experimental material was applied to the other twelve specimens by brushing for 10,000 strokes. Each group was randomly divided into four subgroups according to the period of immersion in phosphate buffer saline (PBS) for 1, 30, 60, and 90 days each. The specimens were sectioned longitudinally and examined with scanning electron microscopy and energy dispersion X-ray spectroscopy. The intratubular crystal were formed in PBS and densely filled the dentinal tubules over time. The crystal formation occurred at a depth of more than 50 μm from the dentin surface. The Ca/P ratio of formed intratubular crystals was 1.68 after 3 months. The experimental material consisting of C2S and C3S with a nanoscale particle size can form hydroxyapatite-like crystals in dentinal tubules in PBS, and there is a possibility of reducing dentin hypersensitivity by blocking the dentinal fluid flow.