Pancreatic cancer tends to be highly resistant to current therapy and remains one of the great challenges in biomedicine with very low 5-year survival rates. Here, we report that zalcitabine, an antiviral drug for human immunodeficiency virus infection, can suppress the growth of primary and immortalized human pancreatic cancer cells through the induction of ferroptosis, an iron-dependent form of regulated cell death. Mechanically, this effect relies on zalcitabine-induced mitochondrial DNA stress, which activates the STING1/TMEM173-mediated DNA sensing pathway, leading to macroautophagy/autophagy-dependent ferroptotic cell death via lipid peroxidation, but not a type I interferon response. Consequently, the genetic and pharmacological inactivation of the autophagy-dependent ferroptosis pathway diminishes the anticancer effects of zalcitabine in cell culture and animal models. Together, these findings not only provide a new approach for pancreatic cancer therapy but also increase our understanding of the interplay between autophagy and DNA damage response in shaping cell death.Abbreviations: ALOX: arachidonate lipoxygenase; ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATM: ATM serine/threonine kinase; ATG: autophagy-related; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; FANCD2: FA complementation group D2; GPX4: glutathione peroxidase 4; IFNA1/IFNα: interferon alpha 1; IFNB1/IFNβ: interferon beta 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MDA: malondialdehyde; mtDNA: mitochondrial DNA; NCOA4: nuclear receptor coactivator 4; PDAC: pancreatic ductal adenocarcinoma; POLG: DNA polymerase gamma, catalytic subunit; qRT-PCR: quantitative polymerase chain reaction; RCD: regulated cell death; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; TFAM: transcription factor A, mitochondrial.