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The aim of this study was to investigate the cytobiological effects of platelet-rich fibrin (PRF) on stem cells from the apical papilla (SCAPs) in vitro and to further explore the underlying molecular mechanisms.SCAPs were isolated from immature third molars. Different concentrations of PRF conditioned medium (one eighth, one quarter, and one half PRF) were prepared. After pretreatment with PRF, the proliferation rate and migration capacity of SCAPs were examined by the Cell Counting Kit-8 assay and wound healing assay, respectively. Alizarin red S staining was performed to examine mineralized nodule formation. Western blot analysis was used to detect the expression of osteo-/odontogenic markers and the extracellular signal–regulated kinase (ERK) pathway in SCAPs. Data were analyzed by one-way analysis of variance. P values <.05 were considered statistically significant.The Cell Counting Kit-8 assay showed that one eighth PRF improved the proliferation rate and the migration capacity of SCAPs (P < .005), whereas one quarter PRF and one half PRF showed no significant difference compared with the control group. The expression of osteo-/odontogenic markers and the capacity to form mineralized nodules of SCAPs were promoted by one eighth PRF and one quarter PRF. In addition, PRF activated ERK signaling, and the ERK inhibitor attenuated PRF-induced osteo-/odontogenesis of SCAPs.PRF improved the osteo-/odontogenic differentiation of SCAPs by activating the ERK pathway; meanwhile, PRF improved the proliferation and migration of SCAPs, and one eighth PRF achieved the most obvious promotion effect. The favorable cytobiological effects of PRF on SCAPs might serve as basis for PRF applications in regenerative endodontic treatment.