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The transcription factor BTB and CNC homology 1 (Bach1) impairs angiogenesis after ischemic injury by suppressing Wnt/β-catenin signaling; however, the specific domains responsible for the antiangiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients, and is the early induction gene after ischemic inuced injury. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-ΔBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) were significantly lower in the AdBach1 group, but not in AdBach1-ΔBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-ΔBTB, in human umbilical-vein endothelial cells (HUVECs) was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF transcriptional activity and mRNA expression in HUVECs. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81-89 of the Bach1 BTB domain and the N-terminal CTNNB1 domain of TCF4. Bach1, but not Bach1-ΔBTB, also copreciptated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy. Funding Statement: This work was supported by the Great Program (91639103 to D. Meng), and General Programs (81670450 and 81873469 to D. Meng, 81570081 and 81770083 to S. Li, 81800234 to L. Jiang, 81572713 to X. Zhi, 81873536 to X. Wang) of the National Natural Science Foundation of China, the National Key Research and Development Program of China (2018YFC1313600 to S. Li). Declaration of Interests: The authors state: "None." Ethics Approval Statement: HUVECs were isolated (12) from the fresh umbilical cord of normal parturients with informed consent. The procedure was approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College. All experimental protocols were approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23).