Osteosarcoma is a primary malignant bone tumor. Although surgery and chemotherapy are the main treatment methods, the overall curative effect remains unsatisfactory. Therefore, there is an urgent need to develop new therapeutic options for osteosarcoma. In this study, the effect and molecular mechanism of osteoblast-derived exosomes on the treatment of osteosarcoma were evaluated. Human primary osteoblasts were cultured to observe the effects of osteoblast-derived exosomes on the osteogenic differentiation of osteosarcoma cells both in vitro and in vivo. Alizarin red staining and alkaline phosphatase detection were used to evaluate the degree of osteogenic differentiation, and immunofluorescence and Western blotting were used to detect protein expression. The results showed that osteoblast-derived exosomes effectively inhibited the proliferation of osteosarcoma cells and promoted their mineralization in vitro. The exosomes also significantly inhibited tumor growth and promoted tumor tissue mineralization in vivo. Osteoblast-derived exosomes upregulated the expression of bone sialoprotein, osteonectin, osteopontin, runt-related transcription factor 2, and Wnt inhibitory factor 1, downregulated the expression of cyclin D1, and suppressed the nuclear accumulation of β-catenin and promoted its phosphorylation in vitro and in vivo. However, these effects were significantly reversed by upregulated gene (URG) 4 overexpression. These findings suggest that osteoblast-derived exosomes could activate the osteogenic differentiation process in osteosarcoma cells and promote their differentiation by targeting the URG4/Wnt signaling pathway.