Despite progress and development in cancer chemotherapeutics and targeted-therapies, the development of drug resistance remains a major obstacle for clinical treatment efficacy. Growing evidence suggests that tumours contain a population of cells known as tumour-initiating cells or cancer stem cells (CSC) that display properties of self-renewal and quiescence that contribute to the maintenance and development of tumours. Additionally, CSCs have been shown to be resistant to conventional clinical therapies through various mechanisms, one of which is the overexpression of antiapoptotic proteins. Our aim is to target CSCs using a novel siRNA delivery method composed of an RNA aptamer, a binding ligand that binds to the EpCAM surface molecule, attached to a siRNA duplex that targets the antiapoptotic protein BIRC5 (survivin) expression. We show that targeting survivin expression in the colorectal cancer cell line HT-29 with the aptamer-siRNA chimera inhibits survivin-mediated drug resistance by affecting the functional properties of self-renewal and tumourigenicity. Sphere-forming assays showed that HT-29 cells maintained high self-renewal capacity when treated with chemotherapeutic agents alone. However, treatment of the cells with the chimera in combination with either 5-fluorouracil or oxaliplatin resulted in a significant decrease in self-renewal by 2.75 and 5.28-fold, respectively. Additionally, combination treatment of HT-29 cells with chimera and 5-fluorouracil cells resulted in weaker tumourigenicity potential when injected into immunocompromised NOD/SCID mice. These data suggest that targeting the CSC population in HT-29 cells limit their self-renewal and tumourigenic capacity, and preliminary in vivo results show promising clinical applications for the aptamer-siRNA chimera.