A hydroxyapatite (HA, Ca10(PO4)6(OH) 2), transformed from a calcium-containing borate glass, has been investigated for its protein adsorption and chromatographic characteristics. Microspheres of the borate glass were transformed into HA by reacting them with a 0.25 M phosphate (K2HPO4) solution for 24 h at 37°C (pH 9.0). The HA microspheres with a diameter of 45-90 µm were hand packed into a steel column (4.6 mm × 80 mm) and used to separate a binary protein mixture of bovine serum albumin (BSA) and lysozyme. HA microspheres, with a diameter <45 µm, were used for separating a protein mixture of BSA, myoglobin, and lysozyme. These microspheres had a diameter that was 20-30 times larger than commercial HA column packing spherical particles, 2-3 µm, but these microspheres had a six times larger surface area and a more uniform spherical shape. These advantages compensated for their larger size and the separation results were comparable to those commercially available HA columns in the separation of the proteins studied. These unique HA microspheres, made from microspheres of a borate glass, are considered to be useful as packing materials for protein separation in chromatography.