AbstractHow cells coordinate cell cycling with cell survival and death remains incompletely understood. Here, we show that cell cycle arrest has a potent suppressive effect on ferroptosis, a form of regulated cell death induced by overwhelming lipid peroxidation at cellular membranes. Mechanistically, cell cycle arrest induces diacylglycerol acyltransferase (DGAT)–dependent lipid droplet formation to sequester excessive polyunsaturated fatty acids (PUFAs) that accumulate in arrested cells in triacylglycerols (TAGs), resulting in ferroptosis suppression. Consequently, DGAT inhibition orchestrates a reshuffling of PUFAs from TAGs to phospholipids and re-sensitizes arrested cells to ferroptosis. We show that some slow-cycling antimitotic drug–resistant cancer cells, such as 5-fluorouracil–resistant cells, have accumulation of lipid droplets and that combined treatment with ferroptosis inducers and DGAT inhibitors effectively suppresses the growth of 5-fluorouracil–resistant tumors by inducing ferroptosis. Together, these results reveal a role for cell cycle arrest in driving ferroptosis resistance and suggest a ferroptosis-inducing therapeutic strategy to target slow-cycling therapy-resistant cancers.