Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia-reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation-dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome-wide CRISPR screening, we found that a mitochondrial Ca uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress-induced ferroptosis. MICU1 is required for mitochondrial Ca increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1-dependent mitochondrial Ca homeostasis-MMP hyperpolarization axis is involved in cold stress-induced lipid peroxidation and ferroptosis. 2+ 2+ 2+