Dental pulp stem cells (DPSCs) are reported to be enriched in stem/progenitor cells, however to the best of our knowledge they have yet to be well documented and characterized. In the present study, in order to characterize DPSCs and the effect of microRNAs (miRs/miRNAs) on DPSC properties, a miRNA array was performed between dental periodontal ligament cells (DPLCs) and DPSCs. The results revealed that miR-224-5p (miR-224) was highly expressed in the DPSCs compared with that in the DPLCs. The transfection of DPSCs with an miR-224 inhibitor impaired cell viability. In addition, miR-224 inhibition significantly promoted cell apoptosis in DPSCscompared with the NC group. In silico analysis and a dual-luciferase reporter assay demonstrated that miR-224 targets the 3'-untranslated region of the Rac family small GTPase 1 (Rac1) gene. miR-224 downregulation resulted in the increased expression of Rac1 in DPSCs compared with DPLCs. Furthermore, miR-224 inhibition caused augmented mitogen-activated protein kinase 8, caspase-3, caspase-9 and Fas ligand expression in DPSC, which may be recovered by Rac1 silencing with transfection with short hairpin RNA-Rac1. Furthermore, Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry indicated that the silencing of Rac1 restored the pro-apoptotic DPSC cell number with miR-224 transfection. Therefore, the results of the present study suggested miR-224 in DPSC serves an important function in protecting cells against apoptosis by downregulating Rac1 expression, and also identified miR-224 as a novel miRNA in regulating the features of DPSC.,Copyright: © Qiao et al.