Calcium (Ca ) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca responses of human dental pulp stem cells (hDPSCs) stimulated with tricalcium silicate-based (TCS-based) dental biomaterials remains largely unexplored. The objective of the present study was to identify and correlate extracellular Ca concentration, intracellular Ca dynamics, pH, cytotoxicity, gene expression and mineralization ability of human dental pulp stem cells (hDPSCs) stimulated with two different TCS-based biomaterials: Biodentine and ProRoot white MTA. The hDPSCs were exposed to the biomaterials, brought in contact with the overlaying medium, with subsequent measurements of extracellular Ca and pH, and intracellular Ca changes. Messenger RNA expression (BGLAP, TGF-β, MMP1 and BMP2), cytotoxicity (MTT and TUNEL) and mineralization potential (Alizarin red and Von Kossa staining) were then evaluated. Biodentine released significantly more Ca in the α-MEM medium than ProRoot WMTA but this had no cytotoxic impact on hDPSCs. The larger Biodentine-linked Ca release resulted in altered intracellular Ca dynamics, which attained a higher maximum amplitude, faster rise time and increased area under the curve of the Ca changes compared to ProRoot WMTA. Experiments with intracellular Ca chelation, demonstrated that the biomaterial-triggered Ca dynamics affected stem cell-related gene expression, cellular differentiation and mineralization potential. In conclusion, biomaterial-specific Ca dynamics in hDPSCs determine differentiation and mineralization outcomes, with increased Ca dynamics enhancing mineralization.