There are two main methods used for dentin hypersensitivity (DH) treatment: dentinal tubule occlusion and blockage of nerve activity. Dentifrices are the most common vehicles for active ingredients used for DH treatment. The aim of this study was to evaluate the efficacy of three toothpastes on dentinal tubule occlusion, mineral acquisition, and dentin hardness. Forty human dentin disks were submerged in 40% citric acid for 30 s and then exposed to tooth brushing for 2 min twice a day for 14 days using three toothpastes: Dontodent Sensitive (group 1), Dr. Wolff’s Biorepair (group 2), and Sensodyne Repair and Protect (group 3). In the control group (group 4), the samples were brushed with water. All of the samples were evaluated using scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and Vickers dentin hardness determination. On SEM images, the degree of dentinal tubule occlusion was assessed using a five-grade scale. The mean score values in groups 1–4 were 3.60 ± 0.69, 2.20 ± 0.91, 2.30 ± 1.16, and 5.00 ± 0.00, significantly higher in study groups when compared to the control group (Kruskal Wallis test p < 0.05). EDX evaluation showed significantly higher calcium and phosphorus concentrations in groups 1 and 3 when compared to control group d. The mean values of Vickers dentin hardness numbers in groups 1–4 were 243.03 ± 10.014, 327.38 ± 56.65, 260.29 ± 37.69, and 225.83 ± 29.93, respectively. No statistically significant results were obtained when comparing the hardness mean values in groups (Kruskal-Wallis statistical test, p = 0.372 > 0.05). All three toothpastes tested demonstrated significant occlusion of dentinal tubules. Dontodent Sensitive and Sensodyne Repair and Protect toothpastes enhanced the calcium and phosphorus content of the dentin surface. None of the toothpastes increased dentin hardness as a result of mineral acquisition.