Adipose-derived stem cells (ADSCs) are considered to be ideal stem cell sources for bone regeneration owing to their ability to differentiate into osteo-like cells. Therefore, they have attracted increasing attention in recent years. Tetrahedral DNA nanostructures (TDNs), a new type of DNA-based biomaterials, have shown great potential for biomedical applications. In the present work, we aimed to investigate the role played by TDNs in osteogenic differentiation and proliferation of ADSCs and tried to explore if the canonical Wnt signal pathway could be the vital biological mechanism driving these cellular responses. Upon exposure to TDNs, ADSCs proliferation and osteogenic differentiation were significantly enhanced, accompanied by the up-regulation of genes correlated with the Wnt/β-catenin pathway. In conclusion, our results indicate that TDNs are crucial regulators of the increase in osteogenic potential and ADSCs proliferation, and this noteworthy discovery could provide a promising novel approach toward ADSCs-based bone defect regeneration.