Background: Portland cement (PC), the base material of mineral trioxide aggregate has been applied in vivo and in vitro studies, and showed promising physical and mechanical properties on several cell lineages but human dental pulp stem cells (DPSCs), known as mesenchymal stem cells with their multipotency. Our study aims to evaluate the cytotoxicity of PC mixing with distilled water (Td) or normal saline (Tn) on DPSCs. Materials and Methods: DPSCs were isolated from pulp tissue and identified before viability assay. DPSCs were treated with the concentration of 100%, 50%, 25%, 12.5% and 6.25% extracts in Td and Tn. Cell viability was evaluate after 24 h, 48 h, 72 h and 7 days treating. Cells were analyzed for cell surface antigen expression by flow cytometry. The pH levels of extracts were detected by pH meter. Cell viability was determined with cell counting kit-8 assay. Results: Viability of Td and Tn showed the general trend that dropped slightly at 24 h, increased at 48 h then showed no statistical differences to control at 72 h. The viability of the concentration of 100% groups dropped gradually from 24 h to 72 h. Cell proliferation was improved in low concentration groups on 48 h and 72 h. On day 7, cell viability showed no statistical difference to control except Tn 50% (110.9%) and Tn 100% (84.0%). Conclusions: PC is probably a potential candidate for the use of pulp therapy, or further, a budding material for pulp regeneration.