Ferroptotic vulnerability is determined by selective subcellular routing of diacylglycerol through a CHPT1–intracellular LCAT axis on Golgi membranes that generates pro-ferroptotic polyunsaturated cholesteryl esters, establishing lipid compartmentalization rather than abundance as the fundamental determinant of ferroptosis and providing therapeutic targets for both cancer suppression and metabolic liver disease.

Ferroptosis is driven by iron-dependent lipid peroxidation, yet how metabolic flux through central lipid pathways is selectively routed towards pro-ferroptotic lipid species remains unclear. Here, through an unbiased chemical–genetic screen targeting core metabolic enzymes, we identify diacylglycerol (DAG) as a licensing lipid intermediate whose pro-ferroptotic activity depends on its intracellular routing. Systematic manipulation of lipolytic flux reveals that ferroptotic vulnerability is not determined by bulk lipolytic output or downstream intermediates, but by the selective channelling of DAG into a distinct metabolic fate. Mechanistically, DAG is selectively routed through a previously unrecognized intracellular metabolic axis composed of choline phosphotransferase 1 (CHPT1) and lecithin–cholesterol acyltransferase (LCAT). We uncover an enzymatically active intracellular pool of LCAT (iLCAT) that cooperates with CHPT1 on Golgi–trans-Golgi network membranes to generate polyunsaturated cholesteryl esters that execute ferroptosis. Functionally, enforced DAG routing through this axis suppresses tumour growth via ferroptosis in vivo, whereas hepatocyte-specific inhibition of the CHPT1–iLCAT axis attenuates lipid peroxidation and disease progression in metabolic dysfunction–associated steatohepatitis. Together, these findings establish subcellular lipid routing, rather than lipid abundance per se, as a fundamental determinant of ferroptotic vulnerability.