Calcium silicate (CS) is envisioned as a good substrate for bone tissue engineering applications because it can provide bioactive ions like Ca2+ and Si4+ to promote bone regeneration. Calcination temperature is a critical factor in determining the crystallinity of CS ceramic, which subsequently influences its degradation and ion release behaviors. To investigate the effect of calcination temperature on the capacity of CS in inducing bone regeneration, CS nanofibers were fabricated via electrospinning of precursor sol-gel and subsequent sintering at 800°C, 1000°C or 1200°C. As the calcination temperature was increased, the obtained CS nanofibers displayed higher crystallinity and slower degradation rate. The CS nanofibers calcined at 800°C (800m) would like to cause high pH (>9) in cell culture medium due to its rapid ion release rate, displaying adverse effect on cell viability. Among all the preparations, it was found the CS nanofibers calcined at 1000°C (1000m) demonstrated the strongest promotion effect on the osteogenic differentiation of bone marrow mesenchymal stromal cells. To facilitate in vivo implantation, the CS nanofibers were shaped into three-dimensional macroporous scaffolds and coated with gelatin to improve their mechanical stability. By implanting the scaffolds into rat calvarial defects, it was confirmed the scaffold made of CS nanofibers calcined at 1000°C was able to enhance new bone formation more efficiently than the scaffolds made of CS nanofibers calcined at 800°C or 1200°C. To summarize, calcination temperature could be an effective and useful tool applied to produce CS bioceramic substrates with improved potential in enhancing osteogenesis by regulating their degradation and bioactive ion release behaviors.