Osteosarcoma is the most common type of aggressive bone cancer. Current treatment strategies include surgical resection, radiation, and chemotherapy. Doxorubicin has been widely used as a chemotherapeutic drug to treat osteosarcoma. However, drug resistance has become a challenge to its use. In this study, p53-wild type U2OS and p53-null MG-63 osteosarcoma-derived cells were used to investigate the mechanism of doxorubicin-induced cytotoxicity. In cell viability assays, doxorubicin effectively induced apoptosis in U2OS cells via the p53 signaling pathway, evidenced by elevated PUMA and p21 protein levels and activated caspase 3 cleavage. In contrast, p53-null MG-63 cells were resistant to doxorubicin-induced apoptosis, while exogenous expression of p53 increased drug sensitivity in those cells. The role of TGF-beta/Smad3 signaling was investigated by using TGF-beta reporter luciferase assays. Doxorubicin was able to induce TGF-beta signal transduction without increasing TGF-beta production in the presence of p53. Knockdown of Smad3 expression by small hairpin RNA (shRNA) showed that Smad3 was required for p53-mediated TGF-beta signaling in response to doxorubicin treatment in U2OS and MG-63 cells. Taken together, these data demonstrate that p53 and TGF-beta/Smad3 signaling pathways are both essential for doxorubicin-induced cytotoxicity in osteosarcoma cells.