Chondroitin sulfate (CS) is the major component of glycosaminoglycan in connective tissue. In this study, we fabricated methacrylated PEGDA/CS-based hydrogels with varying CS concentration (0, 1, 5, and 10%) and investigated them as biomineralizing three-dimensional scaffolds for charged ion binding and depositions. Due to its negative charge from the sulfate group, CS exhibited an osteogenically favorable microenvironment by binding charged ions such as calcium and phosphate. Particularly, ion binding and distribution within negatively charged hydrogel was dependent on CS concentration. Furthermore, CS dependent biomineralizing microenvironment induced osteogenic differentiation of human tonsil-derived mesenchymal stem cells in vitro. Finally, when we transplanted PEGDA/CS-based hydrogel into a critical sized cranial defect model for 8 weeks, 10% CS hydrogel induced effective bone formation with highest bone mineral density. This PEGDA/CS-based biomineralizing hydrogel platform can be utilized for in situ bone formation in addition to being an investigational tool for in vivo bone mineralization and resorption mechanisms.