IFNs have pleiotropic antitumor mechanisms of action. The purpose of this study was to further investigate the effects of IFN-β on the vasculature of human xenografts in immunodeficient mice. We found that continuous, systemic IFN-β delivery, established with liver-targeted adeno-associated virus vectors, led to sustained morphologic and functional changes of the tumor vasculature that were consistent with vessel maturation. These changes included increased smooth muscle cell coverage of tumor vessels, improved intratumoral blood flow, and decreased vessel permeability, tumor interstitial pressure, and intratumoral hypoxia. Although these changes in the tumor vasculature resulted in more efficient tumor perfusion, further tumor growth was restricted, as the mature vasculature seemed to be unable to expand to support further tumor growth. In addition, maturation of the intratumoral vasculature resulted in increased intratumoral penetration of systemically administered chemotherapy. Finally, molecular analysis revealed increased expression by treated tumors of angiopoietin-1, a cytokine known to promote vessel stabilization. Induction of angiopoietin-1 expression in response to IFN-β was broadly observed in different tumor lines but not in those with defects in IFN signaling. In addition, IFN-β–mediated vascular changes were prevented when angiopoietin signaling was blocked with a decoy receptor. Thus, we have identified an alternative approach for achieving sustained vascular remodeling—continuous delivery of IFN-β. In addition to restricting tumor growth by inhibiting further angiogenesis, maturation of the tumor vasculature also improved the efficiency of delivery of adjuvant therapy. These results have significant implications for the planning of combination anticancer therapy. (Mol Cancer Res 2007;5(6):531–42)