Introduction: Dentin hypersensitivity is a common pathology, produced by exposed dental tubules. Objectives: The aim of this study was the evaluation of the effectiveness of experimental toothpaste based on nano-hydroxyapatite (nHAP) obtained from chicken eggshell in the obliteration of dentinal tubules (DTs). Material and methods: Toothpastes with different percentages of nHAP were formulated, including 3%, 7%, and 15% of nHAP, a commercial paste, and a toothpaste without nHAP. Assays were made using healthy premolar samples (n = 50). The pastes were applied and brushed for 7 days after a first erosive cycle, then a second erosive cycle was made at 15 days. Samples were analyzed using scanning electron microscopy and confocal Raman microscopy at three times: after the first erosive cycle (T0), after applications of the pastes (T7), and after the second erosive cycle (T15). Results: The quantitative analysis of the micrographs showed a significant difference between the experimental toothpastes with respect to the controls. Moreover, there was no statistically significant difference between the experimental toothpastes at time T7. By mineral concentration analysis, the 3%-nHAP toothpaste presented a significant difference against conventional toothpaste, when evaluating mineral concentration at 7 days. Conclusions: The experimental paste is effective in obliterating DTs at concentrations of 7 and 15%, in addition to being stable over time.