Reactive intermediates produced during oxidative stress are believed to contribute to the development and progression of a variety of age-related diseases. 4-hydroxy-2-nonenal (HNE), an abundant product of polyunsaturated fatty acid oxidation and decomposition, reacts extensively with DNA and proteins, depletes intracellular glutathione, and alters many cell signaling cascades, including those regulating apoptosis. Using microarray technology, global changes in gene expression were monitored temporally at subcytotoxic and cytotoxic HNE doses in RKO human colorectal carcinoma cells, with the highest level of changes being observed at cytotoxic doses. Gene expression alterations were confirmed using real-time reverse transcription polymerase chain reaction, Western blotting, and transcription factor reporter profiling. Significant alterations were observed in genes regulated by the antioxidant, heat shock, ER stress, and nutrient deprivation responses. Our results demonstrate that HNE simultaneously affects multiple stress signaling pathways, many of which represent potential mechanisms through which HNE alters cellular viability and response to damage.