Objective This article evaluates the biocompatibility and bioactivity of a novel calcium silicate-based sealer by assessing its impact on the viability, mineral deposition, wound closure, and migration activity of human dental pulp stem cells (hDPSCs). Material and Methods AH Plus and AH Plus Bioceramic were pulverized and sterilized according to International Organization for Standardization 10993-5:2009. The hDPSCs were stored raw materials, reaching 80% confluence after passing stem cell marker tests (CD90 98%, CD105 99.7%, CD73 94%, and LinNeg 0.5%) and were at passage 5 to 6 after serum starvation for 24 hours. The study consisted of four groups: AH Plus at concentrations of 1:1 and 1:4, and AH Plus Bioceramic at concentrations of 1:1 and 1:4. Viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay by measuring optical density values, while mineral deposition was evaluated through Alizarin red staining and analyzed with ImageJ software. Migration activity was measured by calculating migration speed and wound closure percentage using a scratch assay at 24 and 72 hours, with results analyzed by ImageJ. Statistical Analysis Viability, migration, and wound closure results were analyzed using one-way analysis of variance. Mineral deposition was analyzed descriptively. Results There were significant differences in the viability and mineral deposition of hDPSCs between calcium silicate-based sealers and epoxy resin-based sealers. Calcium silicate-based sealers showed higher viability and better mineral deposition. The migration speed rate of hDPSCs at 24 hours and wound closure at 24 and 72 hours were significantly greater with the novel calcium silicate-based sealer compared with the epoxy resin-based sealer. Conclusion This study suggests that calcium silicate-based sealers offer advantages over traditional epoxy resin-based sealers, demonstrating superior biocompatibility and bioactivity. These properties may lead to improved clinical outcomes, such as faster healing and fewer posttreatment complications. Further research is needed to explore the full potential of these materials in endodontics. © 2025 Georg Thieme Verlag. All rights reserved.