Purpose: To investigate the influence of sodium hypochlorite (NaOCl) on human dental stem cell proliferation and differentiation. Method: Dental pulp stem cells (DPSCs), periodontal ligament stem cell (PDLSCs), and gingival mesenchymal stem cells (GMSCs) were treated with NaOCl. Cell viability was evaluated with cellular counting kit-8 (CCK8), and cellular adenosine triphosphate (ATP) levels were analyzed by bromodeoxyuridine (BrdU) incorporation and subsequent flow cytometry. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to detect the expressions of differentiation markers. Results: The viability and ATP levels of all three stem cells types were impaired by NaOCl in a concentration-and time-dependent manners. However, the decrease ATP in GMSCs was less than the other two stem cell population (p < 0.05). NaOCl treatment significantly suppressed the proliferation of dental stem cells (p < 0.05). With regard to differentiation marker expression levels, the decrease in Stro-1 was greater in treatment groups when compared to control on Day 7, while increase in levels of dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and osteocalcin (OC) was smaller (p < 0.05). The expressional changes of Stro-1, DSPP, BSP, and OC were more prominent in DPSMs and PDLSCs than in GMSCs. Conclusion: NaOCl dose-dependently impairs the viability, proliferation and differentiation of dental stem cells. Thus, its toxicity to dental stem cells needs to be considered in clinical application.