The cellular processes influenced by consuming polyunsaturated fatty acids remains poorly defined. Within skeletal muscle, a rate-limiting step in fatty acid oxidation is the of movement lipids across the sarcolemmal membrane, and therefore we aimed to determine the effects of consuming Flaxseed oil high in α-linolenic acid (ALA), on plasma membrane lipid composition and the capacity to transport palmitate. Rats fed a diet supplemented with ALA (10%) displayed marked increases in n-3 PUFAs within whole muscle and sarcolemmal membranes (~5-fold), at the apparent expense of arachidonic acid (-50%). These changes coincided with increased sarcolemmal palmitate transport rates (+20%), plasma membrane fatty acid translocase (FAT/CD36; +20%) abundance, skeletal muscle triacylglycerol content (~2-fold) and rates of whole body fat oxidation (~50%). The redistribution of FAT/CD36 to the plasma membrane could not be explained by increased phosphorylation of signaling pathways implicated in regulating FAT/CD36 trafficking events (i.e. phosphorylation of ERK1/2, CaMKII, AMPK, Akt), suggesting the increased n-3 PUFA composition of the plasma membrane influenced FAT/CD36 accumulation. Altogether, the present data provides evidence that a diet supplemented with ALA increases the transport of lipids into resting skeletal muscle in conjunction with increased sarcolemmal n-3 PUFA and FAT/CD36 contents.