RNA interference (RNAi) for controlling gene expression levels using siRNA or miRNA is emerging as an important tool in stem cell biology. However, the conventional methods used to deliver siRNA into stem cells result in significant cytotoxicity and undesirable side-effects. To this end, we have developed a nanotopography-mediated reverse uptake (NanoRU) delivery platform to demonstrate a simple and efficient technique for delivering siRNA into neural stem cells (NSCs). NanoRU consists of a self-assembled silica nanoparticle monolayer coated with extracellular matrix proteins and the desired siRNA. We use this technique to efficiently deliver siRNA against the transcription factor SOX9, which acts as a switch between neuronal and glial fate of NSCs. The knockdown of SOX9 enhanced the neuronal differentiation and decreased the glial differentiation of the NSCs. Our NanoRU platform demonstrates a novel application and the importance of nanotopography-mediated siRNA delivery into stem cells as an effective method for genetic manipulation.