Abstract Vascular endothelial growth factor (VEGF) is a key regulator of both normal and pathological angiogenesis. It plays critical roles in physiological processes such as maintaining vascular stability and promoting skeletal muscle differentiation. Under pathological conditions such as cancer, VEGF serves as a principle proangiogenic factor to facilitate tumor angiogenesis. To date, although many trans-activating factors of VEGF have been well described, transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, which functions to repress the transcription of VEGF via direct binding to the proximal VEGF promoter. In the current study, we have focused on mapping the ZNF24 binding site within the VEGF promoter and exploring its functions in vivo. Utilizing electrophoretic mobility shift assays, we have identified an 11bp fragment of the proximal VEGF promoter as the ZNF24 binding site and show that it is essential for ZNF24-mediated repression. In addition, this fragment can modulate VEGF transcription as a decoy for ZNF24 when introduced into human breast cancer cells. Using transgenic zebrafish as an in vivo model system, we have demonstrated that expression of human ZNF24 induces vascular defects similar to those induced by VEGF knockdown. Importantly, these defects can be rescued by overexpression of VEGF. Analysis of human breast cancer tissues has shown that ZNF24 protein levels are significantly decreased in malignant tissues compared to normal adjacent tissues. Our data support the conclusion that ZNF24 represses VEGF transcription through direct binding to an 11bp fragment of the VEGF proximal promoter and that it may function as a negative regulator of tumor growth by inhibiting tumor angiogenesis. (Supported by NIH P01 CA045548 and The Breast Cancer Research Foundation) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5133. doi:10.1158/1538-7445.AM2011-5133