Oxidative stress is emerging as an important contributor to the pathogenesis of colorectal cancer (CRC), however, the molecular mechanisms by which the disturbed redox balance regulates CRC development remain undefined. Using a liquid chromatography?tandem mass spectrometry-based lipidomics, we found that epoxyketooctadecenoic acid (EKODE), which is a lipid peroxidation product, was among the most dramatically increased lipid molecules in the colon of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC mice. This is, at least in part, due to increased oxidative stress in colon tumors, as assessed by analyzing gene expression of oxidative markers in AOM/DSS-induced CRC mice and human CRC patients in the Cancer Genome Atlas (TCGA) database. Systemic, short-time treatment with low-dose EKODE increased the severity of DSSinduced colitis, caused intestinal barrier dysfunction and enhanced lipopolysaccharide (LPS)/bacterial translocation, and exacerbates the development of AOM/DSS-induced CRC in mice. Furthermore, treatment with EKODE, at nM doses, induced inflammatory responses via JNK-dependent mechanisms in both colon cancer cells and macrophage cells. Overall, these results demonstrate that the lipid peroxidation product EKODE is an important mediator of colonic inflammation and colon tumorigenesis, providing a novel mechanistic linkage between oxidative stress and CRC development.