The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the polyamine pools in the breast cancer cell line L56Br-C1 and induces apoptotic cell death via the mitochondrial pathway. In this study, we have over-expressed the anti-apoptotic protein Bcl-2 in L56Br-C1 cells and investigated the effect of DENSPM treatment. DENSPM-induced cell death was significantly reduced in Bcl-2 over-expressing cells. Bcl-2 over-expression reduced DENSPM-induced release of the pro-apoptotic proteins AIF, cytochrome c, and Smac/DIABLO from the mitochondria. Bcl-2 over-expression reduced the DENSPM-induced activation of caspase-3. Bcl-2 over-expression also prevented DENSPM-induced Bax cleavage and reduction of Bcl-X(L) and survivin levels. The DENSPM-induced activation of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase was reduced by Bcl-2 over-expression, partly preventing polyamine depletion. Thus, Bcl-2 over-expression prevented a number of DENSPM-induced apoptotic effects.