This in vitro study evaluated the effects of dentifrices containing nano-hydroxyapatite (n-HAp) on dentinal tubule occlusion and on mineral deposition. Dentin specimens of ten human teeth were submersed for 30 s in 40% citric acid and then randomly divided into four groups (three study groups and one control group). In the study groups, the dentin samples were exposed to three different n-HAp toothpastes: Karex (Dr. Kurt Wolff GmbH & Co. KG, Bielefeld, Germany), Biorepair Plus Sensitive (Coswell SpA, Bologna, Italy), and Dr. Wolff’s Biorepair (Dr. Kurt Wolff GmbH & Co. KG, Bielefeld, Germany); in the control group no toothpaste was applied. All of the samples were evaluated using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis. In the control group all of the samples showed a frank and wide opening of the dentinal tubules, whereas in the study groups different degrees of tubule closure by mineral depositions were observed. Toothpastes containing n-HAp determined a significant occlusion of dentinal tubules and a significant increase of mineral deposition on the dentin surface. All three tested toothpastes showed similar results regarding the degree of dentinal tubule closure. Varying degrees of differences in calcium, phosphate, carbon, and oxygen ion concentrations among the three tested toothpastes were obtained.