Abstract The objective of this study was to evaluate the effects of diode laser and a desensitizing dentifrice on dentin permeability. Fifty-two root dentin fragments were obtained (5 × 5mm) and treated with 24% EDTA gel. The samples were divided into 4 groups (n = 13): G1, control (no treatment); G2, diode laser (λ = 908 nm, 1.5 W, continuous mode, 20s); G3, application of abrasive dentifrice for 1 minute (Elmex Sensitive Professional (International Gaba); and G4, application of abrasive dentifrice for 1 minute followed by irradiation with diode laser. Ten samples per group were immersed in 2% methylene blue solution for 4h. The specimens were washed, longitudinally sectioned, observed under optical microscopy, photographed and assessed based on the degree of dye leakage. The remaining samples were observed under scanning electron microscopy (SEM). The leakage data were subjected to ANOVA test, followed by Tukey’s t-test (α = 5%). Groups 2, 3 and 4 showed less dye penetration than the control group (p < 0.05), but were similar among each other. SEM images showed that dentinal tubules were open in G1, and fused and occluded in G2. Group 3 showed dentinal tubules that were occluded by the metal ions from the toothpaste. G4 presented similar characteristics to G3, and the presence of fused dentin. The diode laser and the dentifrice were effective in reducing dentinal permeability, and the combination of the two treatments did not show better results than either one used alone.