Ras signaling originates from transient nanoscale compartmentalized regions of the plasma membrane composed of specific proteins and lipids. The highly specific lipid composition of these nanodomains, termed nanoclusters, facilitates effector recruitment and therefore influences signal transduction. This suggests that Ras nanocluster proteolipid composition could represent a novel target for future chemoprevention interventions. There is evidence that consumption of fish oil containing long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic acid (EPA, 20:55,8,11,14,17) and docosahexaenoic acid (DHA, 22:64,7,10,13,16,19) may reduce colon cancer risk in humans, yet the mechanism underlying this effect is unknown. Here, we demonstrate that dietary n-3 PUFA reduce the lateral segregation of cholesterol-dependent and -independent nanoclusters, suppressing phosphatidic acid-dependent oncogenic KRas effector interactions, via their physical incorporation into plasma membrane phospholipids. This results in attenuation of oncogenic Ras-driven colonic hyperproliferation in both Drosophila and murine models. These findings demonstrate the unique properties of dietary n-3 PUFA in the shaping of Ras nanoscale proteolipid complexes and support the emerging role of plasma membrane-targeted therapies.Significance: The influence of dietary long chain n-3 polyunsaturated fatty acids on plasma membrane protein nanoscale organization and KRas signaling supports development of plasma membrane-targeted therapies in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3899/F1.large.jpg Cancer Res; 78(14); 3899-912. 2018 AACR.