Abstract: Abstract There is an increasing interest in novobiocin as it is an modulator of some antitumor agents. Human breast cancer cell lines T47D and MCF7 were used in order to study the antitumor effect and the modulation mechanism of novobiocin. Novobiocin tolerant cell lines were made from T47D and MCF7, and were also used in this research. The results shew that novobiocin inhibited the cells of the breast cancer cell lines, and its effect was dose depandent. The modulation of novobiocin to etoposide (VP16) was different between T47D and MCF7. Novobiocin decreased the cytotoxicity of VP16 in T47D, but increased the cytotoxicity of VP16 in MCF7. The tolerant cell lines were tolerant to novobiocin, but their responds to combine drug treatment were similar to their parental lines.The results suggest that novobiocin is quite significant in clinical chemotherapy.