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There is an increasing demand for biomaterials with skull regeneration for clinical application. However, most of the current skull repair materials still have limitations, such as inadequate sources, poor cell adherence, differentiation, tissue infiltration, and foreign body sensation. Therefore, this study developed porous microsphere-based scaffolds containing mouse embryonic osteoblast precursor cells (MC3T3-E1 cells) and calcitriol (Cal) using gelatin and gelatin/hydroxyapatite through green freeze-crosslinking and freeze-drying. Gelatin was employed to prepare porous microspheres with a particle size of 100-300 μm, containing open pores of 2-70 μm and interconnected paths. Furthermore, the addition of Cal to porous gelatin microsphere-based scaffolds containing MC3T3-E1 cells (PGMSs-MC) and porous gelatin/hydroxyapatite composite microspheres containing MC3T3-E1 cells (HPGMSs-MC) improved their osteoinductivity and cell proliferation and promoted the formation of mature and well-organized bone. The developed Cal-HPGMSs-MC and Cal-PGMSs-MC displayed a good porous structure and cytocompatibility, histocompatibility, osteoconductivity, and osteoinduction. Thus, the designed scaffolds provide a promising prospect for tissue-engineered constructs with skull growth and integration, laying a foundation for further research on the reconstruction of skull defects., (Copyright © 2022. Published by Elsevier B.V.)