Sulfated β-cyclodextrins (S-β-CDs) are useful excipients for improving the solubility of drugs. One such formulation incorporating 5-fluorouracil (5-FU), termed FD(S), showed improved efficacy over 5-FU alone in orthotopic carcinoma xenograft models. S-β-CDs have heparin-like anticoagulant properties, which may have contributed toward the improved antitumor effect of FD(S). S-β-CDs have also been reported to modify a number of processes involved in angiogenesis. Although the anticoagulant nature of S-β-CDs was established, the antiangiogenic properties of S-β-CDs within FD(S) were unknown. The effect of S-β-CD and FD(S) on the proliferation and migration of endothelial cells in live-cell kinetic assays, and the reorganization of human umbilical vein endothelial cells into tubule structures in vitro was assessed. The effects of S-β-CD on angiogenesis in vitro were validated ex vivo using the rat aorta ring assay and the chick embryo chorioallantoic membrane assay. S-β-CD does not alter proliferative endothelial cell sensitivity to 5-FU cytotoxicity. S-β-CD alone and within FD(S) significantly inhibited angiogenesis by impeding endothelial cell migration, resulting in the inhibition of tubule formation and hence new vasculature. In addition to the cytotoxic action of the drug 5-FU, therapeutic inhibition of angiogenesis by S-β-CDs within FD(S) could potentially limit local invasion and metastases. This has important implications for the exploitation of S-β-CDs for drug formulation improvements or for drug delivery of anticancer biologics.