Objectives: The aim of this study was to evaluate the effect of both Nano-hydroxyapatite and conventional size hydroxyapatite in occluding exposed dentinal tubules in comparison to artificial saliva as a control at different aging periods and two different application times.Methods: Twenty-seven intact human upper and lower molar teeth were used in this study. Teeth were sectioned to obtain four dentin blocks of the cervical region from each tooth. Specimens were randomly divided into three main groups according to the tested material, whether Nano-HAP, Conventional HAP or Artificial saliva. Groups were subdivided into subgroups depending on number of applications per day and aging period. Dentinal tubule occlusion was measured using Environmental Scanning Electron Microscope (ESEM) with image analysis. A Three-way ANOVA used to study the variables, followed by Tukey’s post-hoc test for pairwise comparison. A One-way ANOVA was used to compare interaction between variables. Results: A significant increase (P≤0.001) in totally as well as partially occluded dentinal tubules for both Nano-HAP (81.7%+18%) and Conventional HAP (41.4%+44.6%) compared to artificial saliva (21.8%+19.9%). One month period of application showed a significant difference (P≤0.001) for occluding dentinal tubules (81.7%) in comparison with one week (52.5%) and immediate (24.2%). Three daily applications (81.7%) showed a significant difference (P≤0.001) for dentinal tubules occlusion in comparison to two daily applications (64.5%).Conclusion: Tested materials have varying degrees of tubule occlusion in the form of complete and partial occlusion. Nano-hydroxyapatite has a definite and quick potential in tubular occlusion. Three daily applications provide definite tubular occlusion.