Adeno-associated virus (AAV) is a promising vector for systemic delivery of siRNA due to its long-term expression ability without immunogenicity and pathogenicity. However, its broad host tropism and lack of tissue specificity has limited clinical applications such as cancer therapy. Therefore, redirecting the natural tropism of AAV vectors to unique cell surface antigens is an important requirement for in vivo RNAi-based cancer therapy. To exploit the overexpression property of epithelial cell adhesion molecule (EpCAM) in specific cancer types, we here created anti-EpCAM anitibody-conjugated AAV serotype 2 (AAV2) vectors through a streptavidin-biotin bridge. Upon intravenous injection, anti-EpCAM-conjugated AAV2 vectors showed prominent tumor-specific accumulation in EpCAM-positive tumor-bearing mice without undesirable sequestration in liver. In addition, when loaded with transgenes to express shRNA against epidermal growth factor receptor (EGFR), systemically injected anti-EpCAM-conjugated AAV2/shEGFR vectors induced significant downregulation of EGFR expression in tumors, and eventually suppressed tumor growth even at the long dosing interval of two weeks. This in vivo antitumor effect represents the increased infection efficacy of tropism-modified AAV2 vectors and prolonged expression of EGFR shRNA in tumor tissues. Thus, this study suggests the great potential of anti-EpCAM-conjugated AAV2/shEGFR vectors as RNAi-based cancer therapeutics.