In this report, we investigated the role and regulation of forkhead box M1 (FOXM1) in breast cancer and epirubicin resistance. We generated epirubicin-resistant MCF-7 breast carcinoma (MCF-7-EPIR) cells and found FOXM1 protein levels to be higher in MCF-7-EPIR than in MCF-7 cells and that FOXM1 expression is downregulated by epirubicin in MCF-7 but not in MCF-7-EPIR cells. We also established that there is a loss of p53 function in MCF-7-EPIR cells and that epirubicin represses FOXM1 expression at transcription and gene promoter levels through activation of p53 and repression of E2F activity in MCF-7 cells. Using p53-/- mouse embryo fibroblasts, we showed that p53 is important for epirubicin sensitivity. Moreover, transient promoter transfection assays showed that epirubicin and its cellular effectors p53 and E2F1 modulate FOXM1 transcription through an E2F-binding site located within the proximal promoter region. Chromatin immunoprecipitation analysis also revealed that epirubicin treatment increases pRB (retinoblastoma protein) and decreases E2F1 recruitment to the FOXM1 promoter region containing the E2F site. We also found ataxiatelangiectasia mutated (ATM) protein and mRNA to be overexpressed in the resistant MCF-7-EPIR cells compared with MCF-7 cells and that epirubicin could activate ATM to promote E2F activity and FOXM1 expression. Furthermore, inhibition of ATM in U2OS cells with caffeine or depletion of ATM in MCF-7-EPIR with short interfering RNAs can resensitize these resistant cells to epirubicin, resulting in downregulation of E2F1 and FOXM1 expression and cell death. In summary, our data show that ATM and p53 coordinately regulate FOXM1 via E2F to modulate epirubicin response and resistance in breast cancer.