Background: Vital pulp therapy aims to preserve pulp vitality by promoting reparative dentin formation through bioactive materials that support human dental pulp stem cells (hDPSCs) proliferation. Objectives: This study compared the biocompatibility of premixed putty calcium-silicate-based cement (C-SbC) with mineral trioxide aggregate (MTA) on hDPSCs in terms of viability, proliferation, and mineralization potential. Materials and Methods: hDPSCs were divided and cultured in six different groups: MTA, premixed putty C-SbC (Bio-C Repair) at 1:1, 1:2, and 1:4 dilutions, and DMEM as control. Viability and proliferation were assessed using MTT and trypan blue staining after 24 h. Mineralization potential was evaluated qualitatively using Alizarin Red staining after 21 days. Statistical analysis was performed using the one-way analysis of variance and Bonferroni post hoc test (P < 0.05). Results: All the groups showed cell viability with no significant differences in proliferation potential at 24 h. However, cell counts indicated a significant increase in hDPSCs with all concentrations of premixed putty C-SbC compared to MTA (P < 0.05), with the 1:1 concentration (Group 4) showing the highest proliferation. Alizarin red staining revealed more mineral nodules in Group 4, indicating enhanced mineralization. Conclusion: Premixed putty C-SbC, particularly at a 1:1 concentration, demonstrated superior biocompatibility and mineralization potential compared to MTA, making it a promising material for vital pulp therapy. © 2025 Journal of Conservative Dentistry and Endodontics.