Xenogeneic bone procured from the slaughterhouse waste was deproteinated by heat treatment method intended for use as a bone substitute. The effect of heat treatment was investigated by thermal analysis and by physico-chemical methods such as X-ray powder diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy. The heat treatment temperatures for the bovine bone samples were predetermined by thermogravimetric (TG) analysis. The XRD results revealed that the process of heat treatment promoted the crystallinity of bone samples, particularly at 700 and 900°C. There was no secondary phase transformation detected for heat-deproteinated bone except the presence of the hydroxyapatite (HA) phase, which indicated its phase purity even at a higher temperature. The FTIR spectra of raw bone and bone heated at 300°C indicated the presence of organic macromolecules whereas these disappeared in the samples heated at 500, 700 and 900°C, which suggested the removal of antigenic organic matters around 500°C. The same results were also confirmed quantitatively by calculating the amount of collagen using hydroxyproline estimation. There was no significant change in the TG-thermogram of bone heated at 500, 700 and 900°C, which indicated their thermal stability. These findings implied that the heat treated bone at 500°C had properties similar to carbonated HA with low crystallinity, while 700 and 900°C samples had the same with higher crystallinity. As low temperature treatment does not alter morphological and structural properties, we propose that the 500°C heat treated xenogeneic bone may act as an excellent osteogenic bone substitute.