RNA interference, due to its target specificity, may be highly effective as a novel therapeutic modality, but direct delivery of synthetic small interfering RNA still remains a major obstacle for this approach. To induce long-term expression and specific gene silencing, novel delivery vector system is also required. In this study, we have generated an efficient oncolytic adenovirus (Ad)-based short hairpin (shRNA) expression system (Ad-DeltaB7-U6shIL8) against IL-8, a potent proangiogenic factor. To demonstrate IL-8-specificity of this newly engineered Ad-based shRNA, we also manufactured replication-incompetent Ads (Ad-DeltaE1-CMVshIL8 and Ad-DeltaE1-U6shIL8) under the control of the cytomegalovirus (CMV) and U6 promoters, respectively. Ad-DeltaE1-U6shIL8 was highly effective in reducing IL-8 expression, and was much more effective in driving IL-8-specific shRNA than the CMV promoter-driven vector. The reduced IL-8 expression then translated into decreased angiogenesis in vitro as measured by migration, tube formation and rat aortic ring sprouting assays. In addition to its effect on endothelial cells, Ad-DeltaE1-U6shIL8 also effectively suppressed the migration and invasion of cancer cells. In vivo, intratumoral injection of Ad-DeltaB7-U6shIL8 significantly inhibited the growth of Hep3B and A549 human tumor xenografts. Histopathological analysis of Ad-DeltaB7-U6shIL8-treated tumors revealed an increase in apoptotic cells and a reduction in vessel density. Finally, Ad-DeltaB7-U6shIL8 was also shown to inhibit the growth of disseminated MDA-MB-231 breast cancer metastases. Taken together, these findings demonstrate the utility and antitumor effectiveness of oncolytic Ad expressing shRNA against IL-8.