Mesoporous bioactive glass (MBG) as a biodegradable scaffold with a nanostructure has attracted significant attention. However, the in vivo evolution of MBG, which includes in situ degradation, the local effect induced by degradation, and the disposition of degradation products, remains unclear. In this study, we performed in situ labeling and synthesis of an MBG scaffold for the first time using (45)CaCl2. The obtained (45)Ca-MBG scaffolds possessed a mesoporous-macroporous cross-linked structure. These (45)Ca-MBG scaffolds were implanted in critical-sized rat femur defects (3 × 3 mm) for 1 day and for 1, 4, 8, and 12 weeks and analyzed by isotopic quantitative tracing. The results illustrated that the MBG scaffolds gradually degraded over time and persisted at a local level of approximately 9.63% at week 12. This finding suggests that only a very small amount of MBG-released calcium ions may have been transformed into calcium components of the new bone matrix. The research also confirmed that the active ingredients derived from the degradation of MBG scaffolds could actively regulate the mRNA expression levels of osteoblast-related genes in rat bone marrow-derived mesenchymal stem cells (rBMSCs) and promote bone regeneration in vivo. Moreover, through isotopic tracing of the entire body, (45)Ca, which disappeared in situ after implantation, could be detected in the heart, lungs, spleen, kidneys, intestines, and brain via the blood and was mainly accumulated in distal bone tissue, including the radius and cranium. However, (45)Ca radioactivity in the body tissues significantly decreased or disappeared after 12 weeks. Systemic toxicological studies on MBG scaffolds demonstrated the degradation products that spread to major organs did not cause abnormal histopathological changes. The above discoveries comprehensively address crucial issues regarding the application of MBG in vivo, and these findings provide a scientific basis for introducing a material with mesoporous structure into clinical applications.