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Stable and single-dispersed hydroxyapatite (HAP) nanoparticles were synthesized with ultrasonic-assisted method. HAP nanoparticles were characterized by dynamic light scattering, XRD (X-ray diffraction) and TEM (Transmission Electron Microscopy). The effect of HAP nanoparticles on the K562 human myelogcnous leukemia cell line was investigated by MTT assay and cell count test, and the mechanism was studied through the changes of cell cycle and ultrastructure. The results showed that HAP nanoparticles inhibited the proliferation of K562 cells dramatically in vitro. HAP nanoparticles entered the cytoplasm of K562 cells and the cells were arrested at G2/M phase, thus, the cells died directly.