A previous study reported that many supplements have been added to NaF mouthrinses to improve the remineralization potential. Nano-hydroxyapatite (nano-HA) might also be suitable to this purpose because these nano-size particles can penetrate the enamel pores. Moreover, hydroxyapatite is similar to the inorganic component of teeth and is both bioactive and biocompatible. The aim of this study was to evaluate the combined effects of a nano-HA and fluoride mouthrinse on an early caries lesion in human enamel using an in vitro cycle remineralization and treatment model. Forty-eight human enamel specimens, which had a Vickers Hardness Number (VHN) of 25~45 were artificially demineralized for 48h. There were 8 treatment groups (0%, 1%, 5%, 10% nano-HA in distilled water and the same concentrations of nano-HA in a 0.05% NaF solution). The specimens were incubated in an in vitro remineralization model. After immersing the specimens into the treatment and remineralization solution for 12 hours each, the VHN of each specimen was evaluated for total 24 hours. This step was repeated once again for total 48 hours. The enamel surfaces of all specimens were examined by Confocal Laser Scanning Microscopy (CLSM) and SEM. The statistical significance of the data was identified by one-way ANOVA followed by a Duncan’s studentized range test. A p value < 0.05 was considered significant. The results showed that the degree of remineralization, as revealed by the VHN values, was higher in the NaF groups than in the distilled water groups. The VHNs of the remineralized enamel specimens for 48 hours were higher than after the 24 hours treatment. In addition, the level of remineralization increased with increasing concentration of nano-HA and was more pronounced in the NaF groups than the distilled water groups (p<0.05). The CLSM and SEM images nano-sized particles attached to the enamel in the nano-HA treated groups. Nano-HA might play a synergistic role in remineralization with a fluoride mouthrinse. However, more study will be needed to determine the optimal condition of nano-HA and NaF mouthrinse for human use. In conclusion, nano-HA in a 0.05% NaF mouthrinse can help remineralize an early caries lesion.