Abstract Bioactive natural products have led to new drugs or been the inspiration for developing new treatments. Signal transducer and activator of transcription (STAT)3 is a an oncogene that has been implicated in many human cancers. We report that the hirsutinolide natural product, R001, blocks STAT3 DNA-binding activty in vitro. Treatment with R001 of the human triple-negative breast cancer (TNBC) cells, MDA-MB-231 and MDA-MB-468 led to the concurrent inhibition of constitutively-active STAT3 and the expression and activities of glucose-6-phosphate 1-dehydrogenase (G6PD) and thioredoxin reductase (TrxR)1, early suppression of glutathione (GSH), and the induction of reactive oxygen species (ROS), which peaked at 6-24 h post R001-treatment. In the short term, R001-treated TNBC cells showed evidence of pyroptotic cell death. Prolonged treatment of TNBC cells with R001 led to DNA damage and G2/M-phase cell cycle arrest, which are late-stage responses that were preceded by the suppression of ATM-Chk2-Cdc25c pathway, and the induction of p21 and γH2AX phosphorylation by the natural product. Accordingly, R001 suppressed the viable cell numbers, colony formation, growth in 3D-matrigel, and the migration of MDA-MB-231 and MDA-MB-468 cells in vitro. The oral administration of R001 at 5 mg/kg signficantly inhibited growth of human MDA-MB-468 xenografts in mice. Results together show that R001 inhibits STAT3, G6PD and TrxR1 functions that lead to early oxidative stress and pyroptosis and late-stage DNA damage and cell cycle arrest, which collectively cause tumor cell death and tumor growth inhibition in human TNBC models. Citation Format: Yinsong Zhu, Cody F. Dickinson, Justin Yang, Kyrstin Datanagan, Ning Zhai, Peibin Yue, Francisco Lopez-Tapia, Marcus A. Tius, James Turkson. Natural product inhibitor of Stat3, G6PD, and TrxR1 functions induces an early oxidative stress and pyroptosis, and a late-stage DNA damage and cell cycle arrest to block tumor growth in human breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5482.