Despite continued improvements in early detection and therapeutic strategies, chemotherapy-resistance and relapse remains a key obstacle in effective management of metastatic breast cancer. Survivin, a key member of the inhibitor of apoptosis family, has been demonstrated to be a key contributor to the drug-resistant tumourigenic cells and is overexpressed in the MCF-7/ADR doxorubicin-resistant cell line. Through down-regulation of the survivin gene by siRNA transfection, MCF-7/ADR demonstrated an increased doxorubicin sensitivity (4 fold decrease in IC50). Furthermore, the combined treatment of survivin down-regulation followed by doxorubicin treatment showed a significant reduction in tumorigenic capacity (from 11.4% to 3.8%) as determined by the mammosphere formation assay. To develop this strategy into an effective therapeutic, we conjugated the survivin siRNA to an aptamer targeting a tumourigenic cell surface marker, EpCAM, and tested its efficacy in a MCF-7/ADR tumour-bearing mouse model. The aptamer-survivin siRNA fusion, when delivered via intravenous injection, was efficiently endocytosed by the MCF-7/ADR xenograft cells and achieved an in vivo survivin knockdown of ~80%. Mice were treated with aptamer-survivin siRNA, followed by doxorubicin for 3 cycles, and tumours were excised after 9 days. As compared to free doxorubicin, tumourigenic potential was reduced by 85%. A survival study was conducted to compare the efficacy of aptamer-siRNA plus doxorubicin to that of free doxorubicin. Mice bearing MCF-7/ADR orthotopic breast cancer received intravenous injections of aptamer-siRNA or saline (days 1, 3 and 6) and 5mg/kg doxorubicin (days 3, 6, 9). Mice were evaluated over an 11 week period and 90% of animals receiving the combined treatment were still alive as compared to free doxorubicin, where all animals succumbed to tumour-associated mortality by week 9. These results suggest that this tumourigenic cell-targeted delivery system could improve the therapeutic index of doxorubicin and represent a new therapeutic strategy in overcoming chemoresistance in breast cancer.