The ability of the non-steroidal anti-inflammatory drugs (NSAIDs) to prevent colorectal cancer is well established and has been assumed to be mediated through a cyclo-oxygenase (COX) dependent pathway. In this study we demonstrate that naproxen induces cytotoxicity in a COX-2 null human colorectal cancer cell line (HCT-15) and was equipotent in COX expressing cells (DLD-1). Significant decreases in polyamine content (60–45% of control) were observed in both cell lines, with a corresponding increase in the activity of the two major polyamine catabolic enzymes, spermine/spermidine-N1-acetyltransferase (SSAT) and acetylpolyamine oxidase (APAO). Quantitative PCR confirmed a third catabolic enzyme, spermine oxidase (SMO), was also upregulated in both cell lines following exposure to naproxen. These findings indicate that naproxen is capable of inducing changes in polyamine metabolism that could account for its cytotoxicity and possibly also the chemopreventative actions of the NSAIDs, and further confirms a toxicological effect of the NSAIDs independent of COX inhibition.