In this study, we first investigate whether doxorubicin, a daunorubicin analogue that is clinically relevant in breast cancer therapy, leads to nSMase2 upregulation and increased ceramide generation in MCF7 breast cancer cells and uncovers the specific mechanism of nSMase2 transcriptional regulation in response to doxorubicin. This evidence concerns the gene SMPD3 and breast cancer.