Metastatic liver cancer has a very poor prognosis and lacks effective therapy. Anti-angiogenic therapies, which starve tumors of blood supply, have proven to be effective in preclinical models because tumor growth is angiogenesis dependent. However, long-term, high-level, and sustained expression of angiogenesis inhibitors, such as angiostatin, is necessary to prevent dormant tumors from becoming active again. To achieve this objective, we engineered a recombinant adeno-associated virus (AAV) vector encoding mouse angiostatin, an endogenous inhibitor of tumor vascularization. After intraportal delivery of this vector, high-level, stable transgene expression of angiostatin lasting for at least 6 months was observed locally in hepatocytes. Gene transfer of AAV-angiostatin via the portal vein led to significant suppression of the growth of both nodular and metastatic EL-4 lymphoma tumors established in the liver and prolonged the survival time of the mice. The growth of neovessels was inhibited significantly, and extensive apoptosis of tumor cells was observed. The anti-angiogenic activity of angiostatin was independent of vascular endothelial growth factor (VEGF). The AAV-angiostatin viruses did not appear to be toxic to mice, and there was no detectable apoptosis of hepatocytes. In conclusion, these encouraging results warrant future investigation of the use of AAV-mediated anti-angiogenic gene therapy for targeting unresectable liver metastases, especially after surgical removal of primary tumors.