The present study aimed to investigate the effect of Dickkopf-related protein 3 (DKK3) on osteogenic differentiation of rat dental follicle cells (DECs). A PCR array analysis of Wnt pathway activation in DECs identified genes dysregulated by mineral induction. Among them, DKK3expression levels were decreased, and further experiments were conducted to investigate its role in DEC osteogenesis. By comparing DECs grown in normal growth and mineral-induction media for 4 weeks, the present study confirmed that DKK3 was a potential target gene of osteogenesis through reverse transcliption-quantitative polymerase chain reaction (RT-gPCR) and western blotting (WB). A short hairpin RNA (shRNA) was introduced into DECs using a lentiviral vector to inhibit DKK3 expression. An alkaline phosphatase (ALP) activity assay and Alizarin Red staining were performed to observe the DKK3-shRNA DECs. In addition, the osteogenic differentiation of DKK3-shRNA DECs was analyzed by RT-gPCR and WB. In vivo, DKK3-shRNA DECs seeded on hydroxyapatiteiP-tricalcium phosphate (HA/TCP) scaffolds were transplanted into the subcutaneous tissue of mice with severe combined immunodeficiency, followed by hematoxylin-eosin and Masson staining. The results confirmed that DKK3 expression was downregulated during mineral induction in rat DECs. Lentivirus-mediated expression of DKK3 shRNA in DECs promoted calcified-nodule formation, ALP activity and the expression of P-catenin, runt-related transcription factor 2 and osteocalcin, compared with control cells. In vivo, the implanted section presented the majority of newly formed osteoid matrices and collagen, with limited space between the HA/TCP scaffolds and matrices. In conclusion, DKK3 expression negatively regulates the osteogenic differentiation of DECs and, conversely, downregulation of DKK3 may enhance DEC osteogenesis.