Microparticulates are an effective three-dimensional (3D) matrix for the culture of stem cells to be used in tissue engineering of bone. Herein, bioactive calcium phosphate microparticles with an evacuated morphology were prepared, and their potential to support stem cells for bone tissue engineering was addressed. Spherical particles with sizes of hundreds of micrometers were produced using the emulsification method, during which the internal portion was evacuated with the aid of solvent evaporation. The evacuated portion of the microspheres, which is considered to enhance cell population and be replaced with new bone, was found to comprise approximately 65-70% of the total volume. Stem cells derived from human adipose and rat bone marrow were isolated and cultured on the evacuated microspheres. When compared to a two-dimensional culture dish, the 3D spherical substrate provided cells with more space to adhere and populate, which became more evident as the cell seeding quantity increased. Moreover, better cell proliferation was observed on the evacuated microspheres than on the conventional filled microspheres, suggesting that evacuation of the internal part of the microspheres was useful for generating a large cell population. The differentiation of cells cultured on the 3D evacuated microspheres into osteoblasts with appropriate osteogenic cues was also more effective when compared to cells cultured on a two-dimensional dish. When implanted within a rabbit calvarium, the evacuated microspheres induced rapid bone formation at 6 weeks with a typical lamella pattern. Based on the results, the evacuated calcium phosphate microspheres are considered an effective 3D matrix for direct filling of bone defects as well as for bone tissue engineering using stem cells.