Macrophages are critical immune cells for initiating the innate immune response, partly through generation of pro‐inflammatory lipid mediators including eicosanoids. Using LC‐MS lipidomics we have previously characterized the effects of fish oil omega‐3 fatty acid supplementation on TLR4 and purinergic eicosanoid signaling in RAW264.7 and resident macrophages. Supplementation of EPA or DHA leads to inhibition of COX‐1 and COX‐2 arachidonic acid derived pro‐inflammatory prostaglandins, but not inhibition of 5‐LOX in these cells. Consequently, fish oil supplementation can increase production of 5‐LOX AA, EPA and DHA‐derived mono‐hydroxylated metabolites after purinergic stimulation. More recently, we have observed endogenous formation of the pro‐resolution mediator, 15‐epi lipoxin A4, which was dependent on long‐term TLR4 priming followed by P2X7 stimulation with ATP. While mostly trans‐cellular mechanisms have been proposed to explain the switch to pro‐resolution eicosanoid signaling, our finding suggests macrophages can do this through a novel, independent route that requires temporal and combinatorial receptor‐mediated control. We are now characterizing the effects of fish oil omega‐3 supplementation on this mechanism.