Objective: The present double-blind randomized 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. Method 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, and 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 and after 2 and 4 weeks using airblast and tactile tests. In addition, a subjective evaluation using a visual analog scale (VAS) was used. Results: Significantly lower values of cold air sensitivity and tactile sensitivity (P .001) were found for the test group at 2 weeks and 4 weeks. In addition, statistically significantly (P .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 .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 fluoride-free toothpaste as an effective desensitizing agent providing quick relief from symptoms after 2 and 4 weeks.