In striated muscle, EPA and DHA have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment on the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. FAME analysis revealed that both EPA and DHA led to similar but substantial changes in fatty acid profiles. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long chain fatty acids with 5 (EPA treatment) or 6 (DHA treatment) double bonds. As these are typically membrane associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. SILAC based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane associated proteome. We conclude that the EPA specific increase in polyunsaturated long chain fatty acids in the phospholipid fraction is associated with an altered membrane associated proteome and these may be critical events in the metabolic remodelling induced by EPA treatment.