Significance: Phosphatidylethanolamine (PE)-linked arachidonic acid (AA) and adrenic acid (AdA) are well-known substrates for lipid peroxidation, which are indispensable for ferroptosis, an iron-dependent regulated necrosis. However, how cells differentially regulate the intracellular pools of AA and AdA is not fully understood. Here, elongation of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1) are differentially expressed in gastric cancer cells, discriminating the cellular susceptibility to ferroptosis. Biochemical and lipidomics analyses support the hypothesis that ELOVL5 and FADS1 are required to maintain intracellular levels of AA and AdA and promote ferroptosis. Our study highlights the biosynthesis of AA and AdA by ELOVL5 and FADS1 as a critical checkpoint in the ferroptosis pathway.