Epidemiological and preclinical studies demonstrate that consumption of diets high in omega-3 polyunsaturated fatty acids reduces the risk of colon cancer. Inhibition of colon carcinogenesis by omega-3 polyunsaturated fatty acids is mediated through modulation of more than one signaling pathway that alters the expression of genes involved in colon cancer growth. In our earlier studies on global gene expression with cDNA microarrays, we have shown that treatment of CaCo-2 colon cancer cells with docosahexaenoic acid (DHA) down-regulated the prostaglandin family of genes, as well as cyclooxygenase 2 expression and several cell cycle-related genes, whereas it up-regulated caspases 5, 8, 9, and 10 that are associated with apoptosis. It is known that nitric oxide activates the cyclooxygenase 2 enzyme, which plays a pivotal role in the progression of colon cancer via prostaglandin synthesis and angiogenesis. The present study was undertaken to examine the multifaceted role of DHA in the expression of inducible nitric oxide synthase (iNOS) and of related proinflammatory genes, as those have been shown to play a role in tumor progression. In addition, we aimed to identify associated target genes by DNA microarray, reverse transcription-PCR analysis, and cellular localization of iNOS expression in CaCo-2 cells. Results of this study demonstrate that treatment with DHA down-regulates iNOS in parallel with a differential expression and down-regulation of IFNs, cyclic GMP, and nuclear factor kappa B isoforms. More importantly, our findings clearly demonstrate the up-regulation of cyclin-dependent kinase inhibitors p21((Waf1/Cip1)) and p27, differentiation-associated genes such as alkaline phosphatases, and neuronal differentiation factors. These finding strongly suggest that the antitumor activity of DHA may be attributed, at least in part, to an effect on iNOS regulatory genes. In addition, our results indicate the presence of specific gene expression profiles in human colon cancer that can be used as molecular targets for chemopreventive agents.