The study aimed to compare the wear behaviour of human and bovine dentine due to toothbrushing with different relative dentin abrasivity (RDA) toothpastes. Forty human and 40 bovine dentine samples were prepared from bovine lower incisors or human premolars roots, and baseline surface profiles were recorded. The samples were distributed to four groups (each group n = 10 human and 10 bovine samples) and brushed with fluoridated experimental toothpastes with different RDAs (group A: RDA 10, B: RDA 20, C: RDA 50, and D: RDA 100). Toothbrushing was performed in an automatic brushing machine with a brushing frequency of 60 strokes per minute and a brushing force of 2.5 N. After 2, 5, 10, and 25 min of toothbrushing, new surface profiles were recorded, and the dentine wear was calculated with a customized computer programme. The dentine wear of human and bovine dentine within the four groups was compared with unpaired t tests. No statistically significant difference was recorded for the dentine wear of human and bovine samples within the different groups.