Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras, N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.