To investigate whether crosstalk between RUNX2 and miRNAs is involved in tooth eruption regulated by dental follicle cells (DFCs) and the possible molecular mechanism. Blood samples and embedded dental follicles were collected from patients with cleidocranial dysplasia, and RUNX2 gene mutations were analyzed, then RUNX2(+/m) DFCs were isolated and identified. The characteristics of RUNX2(+/m) DFCs were analyzed. The differential expression of miRNAs was detected between the RUNX2(+/m) DFCs and RUNX2(+/+) DFCs by microarray, and target genes were predicted by miRGen. miR-146a was chosen for further investigation, and its effects in DFCs were analyzed by transfecting its mimics and inhibitors, and expression of genes involved in tooth eruption were detected. A novel insertion mutation (c.309_310insTG) of RUNX2 gene was identified which had an effect on the characteristics of DFCs. Compared with the RUNX2(+/+) DFCs, there were 69 microRNAs more than twofold up-regulated and 54 microRNAs more than twofold down-regulated in the RUNX2(+/m) DFCs. Among these, miR-146a decreased significantly in RUNX2(+/m) DFCs, and expression of RUNX2, CSF-1, EGFR, and OPG was significantly altered when miR-146a was overexpressed or inhibited. RUNX2 gene mutation contributes to the characteristic change of DFCs, and the crosstalk between RUNX2 gene and miRNAs may be one of the key regulatory mechanisms of differentiation of DFCs.