RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its short duration of expression. To overcome this shortcoming, we constructed an oncolytic adenovirus (Ad)-based short hairpin RNA (shRNA) expression system (Ad-ΔB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-ΔE1-shVEGF) was also generated. Ad-ΔE1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-ΔB7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-ΔB7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-ΔB7. Ad-ΔB7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-ΔB7-shVEGF was greater than Ad-ΔE1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.