The aim of this in vitro study was to evaluate the tooth whitening effect of toothpaste containing nano-sized hydroxyapatite (HA). There were 3 types of dentifrices, which had a different level of abrasive components. For group 1, a newly developed toothpaste containing Nano-sized hydroxyapatite (Nano-HA) was used. Commercially available toothpastes were used in groups 2 and 3. Group 2 used toothpastes containing silica and multi phosphate. Group 3 used toothpastes containing abrasives with silica and micro-sized HA. In order to simulate 6-months of tooth brushing, the tooth specimens in all 3 groups were placed on a V8 Cross Brushing Machine (Sabri Co. U.S.A.) and brushed 10,000 times with the toothpaste slurries. The tooth specimens were then soaked in artificial saliva for 24 hours to create conditions similar to those encountered in vivo. The whitening efficacy was assessed before and after tooth brushing using the VITA shade scores of Shadeeye-EX NCC Dental Chroma meter (Shofu Co. Japan). All toothpastes produced significant differences in the mean shade change in the teeth before and after tooth brushing (p<0.05). However, there were no significant differences in the change in the shade between each group (p>0.05). Although the new Nano-HA toothpaste did not have superior whitening effects to the commercially available whitening toothpastes, they had a similar whitening efficacy to commercially available whitening toothpastes. These effects might come from the physical properties of Nano HA, which increase the surface dimension more than the Micro-sized HA and have the potential of remineralization.