Glioblastoma (GBM) is the most common and lethal form of primary brain tumor with dismal median and 2-year survivals of 14.5 months and 18%, respectively. The paucity of new therapeutic agents stems from the complex biology of a highly adaptable tumor that uses multiple survival and proliferation mechanisms to circumvent current treatment approaches. Here, we investigated the potency of a new generation of siRNAs to silence gene expression in orthotopic brain tumors generated by transplantation of human glioma stem-like cells in athymic nude mice. We demonstrate that cholesterol-conjugated, nuclease-resistant siRNAs (Chol-hsiRNAs) decrease mRNA and silence luciferase expression by 90% in vitro in GBM neurospheres. Furthermore, Chol-hsiRNAs distribute broadly in brain tumors after a single intratumoral injection, achieving sustained and potent (>45% mRNA and >90% protein) tumor-specific gene silencing. This readily available platform is sequence-independent and can be adapted to target one or more candidate GBM driver genes, providing a straightforward means of modulating GBM biology in vivoMol Cancer Ther; 17(6); 1251-8. ©2018 AACRin vivo.