In regulated cell death, genetically encoded molecular machinery destroys cells. This process is not only essential for organ development and homeostasis, but also leads to pathological diseases. One form of regulated cell death is ferroptosis, which is an iron-dependent oxidative cell death caused by lipid peroxidation. Although inducing ferroptosis is an emerging anticancer strategy, the molecular mechanism underlying tumor resistance to ferroptotic cell death is still unclear. Here, we show that pirin (PIR), an iron-binding nuclear protein, plays a previously unrecognized role in mediating ferroptosis resistance in human pancreatic cancer cells. The transcription factor NFE2L2 mediates the upregulation of PIR during ferroptosis caused by small-molecule compounds (e.g., erastin or RSL3). PIR is a nuclear redox sensor and regulator, and increasing it limits the oxidative damage of DNA and the subsequent cytoplasmic transport and extracellular release of HMGB1. In contrast, the depletion of PIR initiates HMGB1-dependent autophagy by binding to BECN1, and subsequently promotes ferroptosis by activating ACSL4. Consequently, in cell cultures and xenograft mouse models, blocking PIR signaling enhances ferroptosis-mediated tumor growth suppression. Together, these findings provide new insights into the molecular mechanisms of autophagy-dependent ferroptosis. (C) 2020 Elsevier Inc. All rights reserved.