Antitumor imidazoacridinone C-1311 is a DNA-reactive topoisomerase II and FLT3 receptor tyrosine kinase inhibitor. Here, we demonstrate the mechanism of C-1311 inhibitory action on novel targets: hypoxia-inducible factor-1α (HIF-1α), vascular-endothelial growth factor (VEGF), and angiogenesis. In a cell-free system, C-1311 prevented HIF-1α binding to an oligonucleotide encompassing a canonical hypoxia-responsive element (HRE), but did not directly interfere with HIF-1α protein. Whereas C-1311 did not affect HIF-1α transcription, at higher concentrations (10 µM), it decreased HIF-1α protein accumulation. Furthermore, C-1311 potently reduced the transcription of HIF-1-dependent reporter gene as well as endogenous HIF-1 target gene encoding vascular endothelial growth factor. In colon cancer HCT116 and murine melanoma B16/F10 cells, C-1311-induced down-regulation of VEGF, resulted in decreased VEGF protein expression. C-1311 did not alter normoxic VEGF secretion. Consistent with VEGF depletion, C-1311 significantly affected angiogenesis in vivo. A single dose of C-1311 (40 mg/kg), inhibited angiogenesis by 70%, as measured by vascularization of Matrigel plugs in mice. Continuous low C-1311 dosing (8 mg/kg/day for 5 days) gave similar inhibition of angiogenesis (80%), suggesting that low-dose, frequent C-1311 administration may be more effective in terms of reducing toxicity. Anti-angiogenic activity of C-1311 was further demonstrated in an experimental model in which B16/F10 cells were encapsulated in alginate beads and implanted into mice. C-1311 (8 mg/kg/day for 9 days), completely abrogated vascularization of the alginate implants. Our findings indicate that C-1311 is an effective inhibitor of HIF-1α, VEGF, and angiogenesis and provide new perspectives into the mechanism of its anticancer activity.