Abstract Body (Do not enter title and authors here): Background: Homocysteine (Hcy) is a well-established cardiovascular risk factor. Elevated homocysteine, or hyperhomocysteinemia (HHcy), inhibit the growth of endothelial cells and stimulate vascular smooth muscle cell proliferation. However, the mechanisms of HHcy disrupts angiogenesis remain unclear. Here we examine the molecular pathways involved in HHcy-impaired angiogenesis. Method: We generated Tg-hCBS+ Cbs-/- mice by crossing Cbs knockout mice and Tg-hCBS mice where human CBS cDNA was regulated by a zinc-inducible metallothionein promoter. The model exhibited severe HHcy (＞115 μmol/L). We conducted both in vivo and in vitro experiments, including vasculogenesis assays of retinal and heart tissues, aortic ring assay, and tube formation. Retinas were stained with Isolectin B4 and imaged using a confocal microscope. The Human Angiogenesis RT²Profiler™ PCR Array was used to profile 84 key angiogenesis-related genes. Homocysteine metabolites in Cbs mice were measured by HPLC. ChIP-seq data were analyzed using Chip Atlas. Additionally, 91 patients went through coronary angiography were enrolled from the Second Affiliated Hospital of Nanchang University. Participants with CTFC, greater than 27 frames per second were identified as having slow coronary arteries flow (SCF). Results: HHcy caused SCF in patients and impaired vasculogenesis in the retinas of newborn and 12-week-old Tg-hCBS Cbs-/- mice. Capillary density in the hearts of adult Tg-hCBS Cbs-/- mice was reduced. HHcy suppressed tube formation and aortic ring microvessel growth in a concentration-dependent manner. The PCR array revealed significant gene expression changes in Hcy treated human aortic endothelial cells. Notably, genes encoding hepatocyte growth factor (HGF), collagen type IV alpha 3, epiregulin, interferons, and leukocyte cell-derived chemotaxin 1 were downregulated by at least four-fold. In an acute myocardial infarction model, HGF expression was further reduced, and HGF treatment rescued Hcy-inhibited tube formation and microvessel growth. Hcy and S-adenosylhomocysteine levels were negatively correlated with plasma HGF levels. ChIP-seq data identified homocysteine-responsive hypomethylation in ETF, TFII, FOXP3, RXRa, and c-Jun Hcy-hypomethylation Box (-2701/0). Conclusion: HHcy impair angiogenesis via HGF inhibition, which has a rescue effect. Hypomethylation caused by Hcy affects the methylation status of specific CpG sites within the Hcy-hypomethylation box of the HGF gene.