Existing gene delivery technologies using nonviral vectors or physical stimulation are limited by cytotoxicity. Here, we reveal an easy and new method to fabricate silica upright nanosheets, which can be easily scaled up and used for gene delivery. We demonstrate that naked DNA can be transferred into difficult-to-transfect cells (for example, stem cells) by plating cells on silica glass with the upright nanosheets without using any vector. Gene entry is probably achieved through the integrin-FAK-Rho signaling axis, which can be activated by the cytoskeleton rearrangement on nanosheets in a limited time frame (‘transfection window’). Transfecting naked GATA4-binding protein 4 plasmids into stem cells can upregulate the other two important cardiac marker genes myocyte enhancer factor 2C and T-box 5. The transfected mesenchymal stem cells express the cardiac marker proteins at 7 days after transfection, which confirms the success of the innovative gene delivery approach. The vector-free silica nanosheet-induced transfection is simple and effective but faster and safer than the conventional technologies.