Background: Interferon alpha (IFN-α) has antiangiogenic activity, although the underlying mechanism of action is unclear. Because human neuroendocrine (NE) tumors are highly vascularized and sensitive to IFN-α, we investigated whether the therapeutic effects of IFN-α result from an inhibition of angiogenesis mediated by a decrease in vascular endothelial growth factor (VEGF) gene expression. Methods: VEGF gene and protein expression was analyzed in NE tumors by immunohistochemistry and in NE tumor cell lines by quantitative competitive reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). VEGF promoter–reporter gene constructs containing various deletions or mutations and gel shift assays were used to identify minimal promoter requirements and potential transcription factors. A xenograft nude mouse model (five mice per group) was used to determine the effect of IFN-α on tumor growth (NE Bon cells and pancreatic Capan-1 cells) and microvessel density. Liver metastases from eight patients with NE tumors were analyzed for microvessel density, VEGF mRNA content, and VEGF plasma levels before and after initiation of IFN-α therapy. Results: NE tumors and cell lines expressed VEGF mRNA and secreted VEGF protein. In vitro, IFN-α decreased transcription of VEGF gene expression through an Sp1- and/or Sp3-dependent inhibition of VEGF promoter activity. Compared with vehicle treatment in mice, IFN-α inhibited tumor growth by 36% and reduced microvessel density from 56 (95% confidence interval [CI] = 49 to 69) to 37 per ×400 Field (95% CI = 32 to 41, P = .015). Patients with NE tumors had lower VEGF plasma levels and reduced VEGF mRNA levels and microvessel density in liver metastasis biopsy material after IFN-α treatment. Conclusion: IFN-α confers its antitumor activity, at least in part, by its antiangiogenic activity, which results from Sp1- and/or Sp3-mediated inhibition of VEGF gene transcription.