The purpose of this in vitro study was to investigate the migration of dental pulp stem cells (DPSCs) in response to chemotactants and extracellular matrix proteins (EMPs). This DPSC signaling information is needed to help understand tooth regeneration after injury and to develop some future regenerative endodontic therapies.DPSCs were released by trypsinization and plated on transwell filters. The chemotactants were recombinant sphingosine-1-phosphate (S1P), fibroblast growth factor (FGF), epidermal growth factor (EGF), or transforming growth factor beta-1 (TGF-β1), and the EMPs were collagen-1, collagen-IV, laminin, and fibronectin. Data were analyzed by using analysis of variance (ANOVA) statistical tests for cell migration.S1P induced more vigorous DPSC migration in comparison with the other TGF- β1, FGF, or EFG chemotactants (ANOVA, P < .05). Laminin induced more vigorous DPSC migration in comparison with the other EMPs (ANOVA, P < .05).The EMPs, particularly laminin, and chemotactants, particularly S1P and TGF-β1, were found to be important promoters of DPSC migration. The interplay between the EMPs, blood lipid, serum, and chemotactants suggests that the migration of DPSC is highly regulated. Specific chemotactants and EMPs might mediate the process of pulp-dentin regeneration after tooth injury, and they could be used as part of regenerative endodontic therapy.