The synthesis of nanohydroxyapatite from cuttlefish bone (Sepia sp.) has been done by using High Energy Milling (HEM) and its characterization in vitro as bone repair. This study aimed to determine the effect of the milling process on microscopic properties and mechanical properties of nano-HA through XRD, TEM, and compressive strength tests. The hydroxyapatite (HA) used in this study consisted of 1M CaCO3 from aragonite which was extracted from cuttlefish lamella bone (Sepia sp.) and 0.6 M NH4H2PO4, which was hydrothermally processed at 200°C for 12 h and then sintered at 900°C for 1h. Parameter milling includes the variation of milling time, i.e., 3 h, 6 h, and 9 h at rotational speed of 350 rpm. An increase in milling time causes a decrease in HA particle size. This is shown from the results of TEM at the milling time of 9 h with the smallest size up to 65 nm. The result of cell viability test showed that all samples are not toxic with cell viability value of >80%. The milling time of 9 h was an optimum condition with a compressive strength of 4.35952 MPa that can be applied to cancellous bone.