This study investigated the effect of toothbrush stiffness and dentifrice slurry abrasivity on the development and progression of simulated non-carious cervical lesions (NCCLs). Human maxillary premolars were allocated to 12 groups generated by the association between toothbrushes, soft, medium, and hard stiffness, and simulated dentifrice slurries, lower, medium, and higher; deionized water (DI) served as negative control. Teeth were mounted on acrylic blocks, and their root surfaces partially covered with acrylic resin to simulate gingiva, leaving a 2-mm area apical to the cemento-enamel junction exposed to toothbrushing. Specimens were brushed with the test slurries for 35,000 and 65,000 double strokes. Impressions taken at baseline and after both brushing periods were scanned by a 3D optical profilometer. Dentin volume loss (mm3) was calculated by image subtraction. Data were analyzed using three-way ANOVA and Fisher's PLSD tests. All toothbrushes caused higher volume loss when associated to higher abrasive slurry, compared to medium- and lower-abrasive slurries. Medium caused more volume loss than lower-abrasive slurry, which led to more volume loss than DI. Hard and medium toothbrushes were not different when used with medium- or higher-abrasive slurries. There were no differences among toothbrushes when used with DI and lower-abrasive slurry. Overall, 35,000 brushing strokes resulted in significantly less volume loss than 65,000. Toothbrush stiffness was an important factor on NCCL development, especially when brushing with medium- and higher-abrasive slurries. Medium and hard toothbrushes associated with medium- and high-abrasive toothpastes can yield more severe NCCLs.