Abstract The aim of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of dental follicle cells (DFCs) in vitro and on the regenerative effects of DFC-OsteoBoneTM complexes in vivo. DFCs were isolated and characterized. In the in vitro study, DFCs were cultured in an osteogenic medium in the presence or absence of LIPUS. The expression levels of ALP, Runx2, OSX, and COL-I mRNA were analyzed using real-time polymerase chain reaction (RT-PCR) on day 7. Alizarin red staining was performed on day 21. The state of the growth of the DFCs that were seeded on the scaffold at 3, 5, 7, and 9 days was detected by using a scanning electron microscope. In our in vivo study, 9 healthy nude mice randomly underwent subcutaneous transplantation surgery in one of three groups: group A, empty scaffold; group B, DFCs + scaffold; and group C, DFCs + scaffold + LIPUS. After 8 weeks of implantation, a histological analysis was performed by HE and Mason staining. Our results indicate that LIPUS promotes the osteogenic differentiation of DFCs by increasing the expression of the ALP, Runx2, OSX, and COL-I genes and the formation of mineralized nodules. The cells can adhere and grow on the scaffolds and grow best at 9 days. The HE and Mason staining results showed that more cells, fibrous tissue and blood vessels could be observed in the DFCs + scaffold + LIPUS group than in the other groups. LIPUS could promote the osteogenic differentiation of DFCs in vitro and promote tissue regeneration in a DFCs-scaffold complex in vivo. Further studies should be conducted to explore the underlying mechanisms of LIPUS.