The dependency of cancer cells on iron increases their susceptibility to ferroptosis, thus providing new opportunities for patients with treatment-resistant tumors. However, we show that lipid peroxidation, a hallmark of ferroptosis, was found in various areas of patient samples, indicating the potential resistance of ferroptosis. Using whole deubiquitinases (DUBs) sgRNA screening, we found that loss of ZRANB1 confers cancer cell resistance to ferroptosis. Intriguingly, functional studies revealed that ZRANB1 ubiquitinates and represses SLC7A11 expression as an E3 ubiquitin ligase and that ZRANB1 inhibits glutathione (GSH) synthesis through SLC7A11 degradation, leading to elevated lipid peroxidation and ferroptosis. Deletion of the region (residues 463-584) abolishes the E3 activity of ZRANB1. Moreover, we show that ZRANB1 has lower expression in tumors, which is positively correlated with lipid peroxidation. Collectively, our results demonstrate the role of ZRANB1 in ferroptosis resistance and unveil mechanisms involving modulation of E3 ligase activity through an unconventional catalytic domain.© 2023 Huang et al.