Objectives: The present randomized double blind clinical trial aimed to compare the efficacy in reducing dentin hypersensitivity of a dentifrice formulation containing nano-Hydroxyapatite with a fluoride dentifrice and a placebo. Methods and Materials: 105 subjects were recruited to participate in the study. A computer-generated random table with blocking to one of the three study treatments was used in order to have 35 subjects per group: 1) nano-Hydroxyapatite 15% toothpaste fluoride free; 2) fluoride toothpaste; 3) placebo. Groups 1, 2, 3 were instructed to brush their teeth for 2 minutes twice a day with the provided toothpaste. The participant’s dentin hypersensitivity was evaluated at baseline, after 2 and 4 weeks using airblast and tactile tests. In addition a subjective evaluation using a visual analogue scale was used. Results: Significant lower values of cold air sensitivity and tactile sensitivity (p< 0.001) were found for the test group at 2 weeks and 4 weeks. In addition statistically significant (p< 0.001) lower values of sensitivity were reported for group 1 compared to groups 2 and 3 at 2 and 4 weeks respectively. The VAS scores were significantly lower (p< 0.001) in the test group at 2 and 4 weeks compared to baseline and to the control groups. Conclusion: The findings of the present study encourage the application of nano-Hydroxyapatite in tooth paste fluoride free as an effective desensitizing agent providing quick relief from symptoms after 2 and 4 weeks.