Successful short interfering RNA (siRNA)-based therapy for cancers depends on functional siRNA delivery specific to tumors. In our previous report, we have shown systemic siRNA delivery specific to human prostate cancer cell line PC-3 subcutaneous tumors in nude mice by atelocollagen, a collagen derivative, for formulating a complex with siRNA. We used an siRNA for human Bcl-xL as a model target. In the present study, we examined the antitumor effect on PC-3 orthotopic tumors in nude mice, as these tumors resemble the human clinical situation. The systemic intravenous administration of the complex (siRNA, 50 μg/shot) significantly reduced Bcl-xL expression and induced apoptosis in the tumors, and suppressed their growth. Liver metastasis was also inhibited in the orthotopic model. We successfully showed tumor-specific accumulation of the siRNA by Cy3-labeled siRNA and the direct quantification of the siRNA via reverse-phase high-performance liquid chromatography. The tumor-specific delivery was achieved by the enhanced permeability and retention effect, which is characteristic of macromolecular drugs. The high expression of vascular endothelial growth factor-A in the tumors provided adequate conditions to promote the permeability in the tumors, and to finally form the enhanced permeability and retention effect. In conclusion, our siRNA delivery is specific to the PC-3 orthotopic tumors in nude mice, and is practically feasible to treat tumors.