Despite current progress in early detection and anticancer treatment strategies, drug resistance in cancer remains a major obstacle for an effective clinical outcome. Evidence suggest that a population of tumour cells are not only resistant to conventional chemotherapy, but also possess stem cell properties that contribute to tumour growth and relapse known as cancer stem cells or tumour-imitating cells. Survivin, a key member of the inhibitor of apoptosis family, has been shown to be associated with drug-resistance and survival, making it an attractive target for siRNA-based therapy. However, the delivery of siRNAs in vivo can be difficult as they are susceptible to degradation and systemic clearance, and lacks the binding specificity to target the cancer cells. To specifically target the cancer stem cells, we conjugated a survivin-specific siRNA to an aptamer targeting a tumourigenic cell surface marker, EpCAM, and tested its efficacy in both in vitro and in vivo HT-29 tumour-bearing mouse models. The aptamer-siRNA chimera showed effective binding and internalization in the EpCAM-expressing HT-29 cells, and successfully resulted in survivin knockdown by 66.3% ± 13.8. Additionally, the resulting survivin knockdown coupled with 5-flouruoacil treatment showed significant reduction in self-renewal and tumourigenic potential as determined by sphere-formation assay (60.3% ± 0.0196) and xenograft limited dilution assay. These results suggest that the aptamer-siRNA chimera is an effective tool to target cancer stem cells, and represents a new strategy in overcoming drug-resistance in cancer as a supplementary drug with today’s conventional chemotherapy.