c-Myc is a ubiquitous transcription factor that regulates a wide variety of genes involved in the control of cell proliferation, differentiation and apoptosis. It was demonstrated that over-expressions of c-Myc and Nbs1 attenuated the ability of doxorubicin in reduction of colony formation in U2OS cells, and it was found that this effect of c-Myc was associated with Nbs1. It can be confirmed that Nbs1 is a target gene of c-Myc. Chromatin immunoprecipitation assays reveal that c-Myc recruited the histone acetyltransferase p300 complex to the promoter region of Nbs1 gene, resulting in an increased level of acetylated histone H4. Furthermore, it was found that the two proximal E-box elements located in Nbs1 promoter region were essential for the c-Myc binding. Thus, it was concluded that c-Myc attenuates the effect of doxorubicin partially through regulating Nbs1, implicating its roles in repair of DNA breakage induced by doxorubicin.