The aim of this study was to evaluate a new treatment modality for the occlusion of dentinal tubules (DTs) via the combination of 10.6 µm carbon dioxide (CO2) laser and nanoparticle hydroxyapatite paste (n-HAp). Forty-six sound human molars were used in the current experiment. Ten of the molars were used to assess the temperature elevation during lasing. Thirty were evaluated for dentinal permeability test, subdivided into 3 groups: the control group (C), laser only (L-), and laser plus n-HAp (L+). Six samples, two per group, were used for surface and cross section morphology, evaluated through scanning electron microscope (SEM). The temperature measurement results showed that the maximum temperature increase was 3.2 °C. Morphologically groups (L-) and (L+) presented narrower DTs, and almost a complete occlusion of the dentinal tubules for group (L+) was found. The Kruskal-Wallis nonparametric test for permeability test data showed statistical differences between the groups (P < 0.05). For intergroup comparison all groups were statistically different from each other, with group (L+) showing significant less dye penetration than the control group. We concluded that CO2 laser in moderate power density combined with n-HAp seems to be a good treatment modality for reducing the permeability of dentin.