Bone defects are still an unsolved clinical issue that must be overcome. Carbon dots have shown very promising effects in biological therapy. In the current study, we explored their effects on osteogenesis. Furthermore, we revealed the mechanisms in order to develop novel therapeutic approaches to manage the bone defect. For this study, ascorbic acid carbon dots (CDs) were created by a one-step microwave-assisted method. Results showed that the CDs effectively enhanced matrix mineralization, promoted osteogenic differentiation in vitro, and promoted new bone regeneration in the skull defect model in vivo. Furthermore, our data demonstrated that the ER stress and PERK-eIF2α-ATF4 pathway were activated by the CD-induced increase in intracellular calcium. Taken together, our findings suggest that the PERK pathway plays a critical role in CD-induced osteogenic differentiation, and the CDs created herein have the potential to be used to repair bone defects in clinical practice.