Doxorubicin plays a major role in the treatment of osteosarcoma disorders. The Notch signaling pathway exerts various biological functions, including cell proliferation, differentiation, and apoptosis. In the present study, we investigated the effects of different doses of doxorubicin on proliferation and apoptosis of osteosarcoma cells with or without Notch signaling. Results found that cellular viability was downregulated while caspase 3 activity and expression were promoted in osteosarcoma cells following treatment with various doses of doxorubicin for 24, 48, and 72 h, and the effects showed a dose- and time-dependent manner. Furthermore, it was found that various doses of doxorubicin activated the Notch signaling pathway, shown by the elevated expression of Notch target genes NOTCH1, HEY1, HES1, AND HES5. It was further proved that, after small interfering RNA (siRNA)-mediated knockdown of Notch, the effects of doxorubicin on the viability and apoptosis of osteosarcoma cells were significantly reduced. It was indicated that doxorubicin treatment reduced the proliferation and promoted the apoptosis of osteosarcoma cells, and this effect was mediated by the Notch signaling pathway.