MicroRNAs (miRNAs) exhibit strong potential clinical application owing to their extensive regulation and flexible delivery properties. MicroRNA-31 (miR-31) is an evolutionarily conserved miRNA expressed during tooth development, and it is highly expressed in mouse incisor epithelium. The specific role of miR-31 in odontogenesis has not been elucidated comprehensively, and the aim of the present study was to investigate its activity. Our results showed that miR-31 suppressed LS8 cell proliferation by inhibiting the cell cycle at the G1/S transition. Mutation of Special AT-rich sequence-binding protein 2 (SATB2) gene is responsible for human SATB2-associated syndrome (SAS), which is often accompanied by dental abnormities. Here, it was identified as a direct target of miR-31 in LS8 cells and a promoter of cell proliferation. The expression and distribution of SATB2 in mouse molars and incisors were explored using immunofluorescence, which showed strong signals in the nuclei of incisor epithelial cells and weak signals in the cytoplasm of molar epithelial cells. Moreover, rescue experiments demonstrated that Satb2 could mitigate the inhibitory effect of miR-31 on cell proliferation by promoting the expression of CDK4. Collectively, our results suggested that miR-31 regulates dental epithelial cell proliferation by targeting Satb2, highlighting the biological importance of miR-31 in odontogenesis. (C) 2020 Elsevier Inc. All rights reserved.