ObjectivesTo investigate the effect of toothpaste abrasive level on the progression of non-carious cervical lesions (NCCLs) using 3D-image subtraction. MethodsUpper first premolars were allocated into seven groups (n = 16) of toothpaste/abrasive slurries: A-Zeodent113/5%, B-Zeodent124/10%, C-Zeodent103/15%, D-Sensodyne Pronamel, E-Crest Cavity-Protection, F-Crest Pro-Health-Whitening, and G-Deionized water (DIW). Teeth were mounted on acrylic blocks, and their root surfaces covered with acrylic resin, except for 2-mm near the cemento-enamel junction that was exposed to toothbrushing. Specimens were brushed with the slurries for 5000-, 15,000-, 35,000- and 65,000-strokes. Impressions were taken at baseline and after each brushing time, and then scanned by a 3D optical profilometer. Dentine volume loss was calculated by image subtraction software and subjected to mixed-model ANOVA and multiple comparison tests (α = 0.05). ResultsNo significant differences among slurries were observed at 5000 and 15,000. At 35,000, F showed higher loss than all other groups except C, which did not differ from the others. At 65,000, F (4.19 ± 3.29 mm3) showed the highest loss, followed by C (2.33 ± 1.47 mm3), which differed from all the other groups except B (1.85 ± 0.91 mm3). Groups B, A (1.35 ± 0.65 mm3), D (1.17 ± 0.48 mm3), E (1.40 ± 0.68 mm3) and G (1.12 ± 0.73 mm3) did not differ from each other. Groups F and C showed significant increase of volume loss starting at 35,000, while B, A, D and E only at 65,000; no increase loss was observed for G. Conclusions3D-image subtraction was able to quantify and differentiate tooth loss, but only at advanced stages. The progression of NCCLs was more evident and faster for highly abrasive slurries. Clinical significanceUpon root dentin exposure, brushing with lower abrasive dentifrices is advisable to reduce the risk for NCCLs development.