Background: Nanohydroxyapatite (nHAP) exhibit anti-proliferative effects on various cancer cells. However, to date, there are only a few studies on the radiosensitization effect of nHAP. The present study aimed to investigate the possible enhancement of the radiosensitization effect of nHAP on human breast adenocarcinoma cancer (MCF-7) and fibroblast.Methods: nHAP was extracted from fish scales using the thermal alkaline method and characterized at Babol University of Medical Sciences (Babol, Iran) in 2017. The anti-proliferative and the radiosensitization effects of nHAP were investigated by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT), clonogenic assay, and apoptosis assay. MCF-7 cells and fibroblasts were incubated with different concentrations of nHAP and at different periods. The MTT solution was added and the absorbance was measured at 570 nm. The MCF-7 cells were exposed to 0, 1.5, 3.5, and 5 Gy X-ray irradiation and incubated for 10-14 days. The data were compared using the one-way analysis of variance (ANOVA) followed by the post hoc tests (Tukey’s method). Results: The results showed that nHAP significantly inhibited the growth of MCF-7 cells compared with controls (P<0.001), but the difference was not statistically significant for fibroblasts (P=0.686 at 400 μg/mL at 72 hours). After 48 hours, the proliferation of MCF-7 cells and fibroblasts was inhibited by about 81% and 34% at 400 μg/mL concentration, respectively. The radiosensitization enhancement factor for MCF-7 cells and fibroblasts at a dose of 3.5 Gy and 100 μg/mL concentration were 1.87 and 1.3, respectively. Conclusion: nHAP can be considered as a breast cancer radiosensitization agent with limited damage to the surrounding healthy tissue.