Hydroxyapatite is one of calcium phosphate phase and widely use in medical and dental application. Hydroxyapatite could be synthesized from natural calcium source. Cuttlefish bone waste are mainly composed of calcium carbonate. This paper, hydroxyapatite was synthesized from cuttlefish bone by high energy ball milling technique. The cuttlefish bone was heated at different temperature from 200 to 1300 ºC. The heated cuttlefish bones at different temperature were mixed with di ammonium hydrogen orthophosphate ratio 5:3 moles and added distil water 25 ml. The solution was milled at different time from 5 to 120 min. The structure, morphology and function group of cuttlefish bone and hydroxyapatite samples were investigated with X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), respectively. The results show that the natural phase of cuttlefish bone was aragonite phase of calcium carbonate (CaCO3) and changed to pure calcite phase after heated at 500 ºC. The calcite phase of cuttlefish bone was completely transformed to calcium oxide (CaO) after heated at 900 ºC. For hydroxyapatite synthesizing show that the samples were milled for 5 minutes not only hydroxyapatite phase appeared but also remained initial phase. The pure hydroxyapatite phase appeared that the both aragonite and lime phase precursors were milled for 60 minutes. From experiment, the cuttlefish bone before and after heated can be use a source of calcium for hydroxyapatite synthesizing by high energy ball milling technique.