Tooth regeneration through stem cell-based therapy is a promising treatment for tooth decay and loss. Human dental pulp stem cells (hDPSCs) have been widely identified as the stem cells with the most potential for tooth tissue regeneration. However, the culture of hDPSCs invitro for tissue engineering is challenging, as cells may proliferate slowly or/and differentiate poorly invivo. Dynamic three‑dimensional (3D) simulated microgravity (SMG) created using the rotary cell culture system is considered to an effective tool, which contributes to several cell functions. Thus, the present study aimed to investigate the effect of dynamic 3D SMG culture on the proliferation and odontogenic differentiation abilities of hDPSCs in poly (lactic‑co‑glycolic acid) (PLGA) scaffolds in nude mice. The hDPSCs on PLGA scaffolds were maintained separately in the 3D SMG culture system and static 3D cultures with osteogenic medium for 7days invitro. Subsequently, the cell‑PLGA complexes were implanted subcutaneously on the backs of nude mice for 4weeks. The results of histological and immunohistochemical examinations of Ki‑67, typeI collagen, dentin sialoprotein and DMP‑1 indicated that the proliferation and odontogenic differentiation abilities of the hDPSCs prepared in the 3DSMG culture system were higher, compared with those prepared in the static culture system. These findings suggested that dynamic 3DSMG culture likely contributes to tissue engineering by improving the proliferation and odontogenic differentiation abilities of hDPSCs invivo.