Polyamines have been shown to play a role in the growth and survival of several solid tumors, including colorectal cancer. We identified the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as being one of the most highly inducible genes in two DNA microarray screens to identify novel determinants of response to chemotherapeutic agents in colorectal cancer. SSAT was shown to be inducible in response to 5-fluorouracil (5-FU) or oxaliplatin in parental and drug-resistant HCT116 cell lines. It was also shown that SSAT mRNA was up-regulated in response to 5-FU or oxaliplatin in a panel of six colorectal cancer cell lines. The polyamine analogue N(1),N(11)-diethylnorspermine (DENSpm) depletes polyamine pools and potently induces SSAT. We evaluated the effect of combining DENSpm with chemotherapeutic agents in HCT116 p53(+/+) cells and in HCT116 drug-resistant daughter cell lines. Western blot analyses showed that SSAT protein expression was dramatically enhanced when DENSpm was combined with oxaliplatin or 5-FU in HCT116 p53(+/+) cells. Using cell viability assays and flow cytometry, synergistic induction of cell death was observed following cotreatment of HCT116 p53(+/+) cells with DENSpm and each chemotherapeutic agent. Of note, this combined therapy increased the chemosensitivity of cells rendered resistant to each of these chemotherapeutic agents. Small interfering RNA-mediated down-regulation of SSAT resulted in loss of synergy between DENSpm and these agents. These results show that SSAT plays an important role in regulating cell death following combined cytotoxic drug and DENSpm treatment. Furthermore, DENSpm sensitizes both sensitive and resistant cells to chemotherapeutic agents. Taken together, these results suggest that SSAT may be an important target for therapeutic intervention in colorectal cancer.