Abstract: Objective：To obtain human dental follicle cells with RUNX2 gene mutation. Methods：Patients with cleidocranial dysplasia were collected, and RUNX2 gene mutation was identified. The dental follicle cells were cultured from unerupted dental follicle in vitro, and the same mutation site was identified. Results：The novel insertion mutation was identified as c.309_310insTG in exon 2 of RUNX2 gene by genome detection with whole blood, and the dental follicle cells with the same mutation were successfully cultured and identified. The expression of RUNX2 was decreased in RUNX2+/m dental follicle cells. Conclusions：Human dental follicle cells with RUNX2 mutation were successfully cultured and identified, and it contributes to the further function study of RUNX2 gene during tooth eruption.