Advanced cancers acquire resistance to chemotherapy, and this results in treatment failure. The cellular mechanisms of chemotherapy resistance are not well understood. Here, for the first time, we show that ceramide contributes to cellular resistance to doxorubicin through up‐regulating the gene expression of glucosylceramide synthase (GCS). Ceramide, a cellular lipid messenger, modulates doxorubicin‐induced cell death. GCS catalyzes ceramide glycosylation, converting ceramide to glucosylceramide; this process hastens ceramide clearance and limits ceramide‐induced apoptosis. In the present study, we evaluated the role of the GCS gene in doxorubicin resistance using several paired wild‐type and drug‐resistant (doxorubicin‐selected) cancer cell lines, including breast, ovary, cervical, and colon. GCS was overexpressed in all drug‐resistant counterparts, and suppressing GCS overexpression using antisense oligonucleotide restored doxorubicin sensitivity. Characterizing the effect mechanism showed that doxorubicin exposure increased ceramide levels, enhanced GCS expression, and imparted cellular resistance. Exogenous C₆‐ceramide and sphingomyelinase treatments mimicked the influence of doxorubicin on GCS, activating the GCS promoter and upregulating GCS gene expression. Fumonisin B₁, an inhibitor of ceramide synthesis, significantly suppressed doxorubicin‐up‐regulated GCS expression. Promoter truncation, point mutation, gel‐shift, and protein‐DNA ELISA analysis showed that transcription factor Sp1 was essential for ceramide‐induced GCS up‐regulation. These data indicate that ceramide‐governed GCS gene expression drives cellular resistance to doxorubicin.—Liu, Y.‐Y., Yu, J. Y., Yin, D., Patwardhan, G. A., Gupta, V., Hirabayashi, Y., Holleran, W. M., Giuliano, A. E., Jazwinski, S. M., Gouaze‐Andersson, V., Consoli, D. P., Cabot, M. C. A role for ceramide in driving cancer cell resistance to doxorubicin. FASEB J. 22, 2541–2551 (2008)