An efficient nonviral platform for high-throughput and subcellular precision targeted intracellular delivery of nucleic acids in cell culture based on magnetic nanospears is reported. These magnetic nanospears are made of Au/Ni/Si (∼5 μm in length with tip diameters <50 nm) and fabricated by nanosphere lithography and metal deposition. A magnet is used to direct the mechanical motion of a single nanospear, enabling precise control of position and three-dimensional rotation. These nanospears were further functionalized with enhanced green fluorescent protein (eGFP)-expression plasmids via a layer-by-layer approach before release from the underlying silicon substrate. Plasmid functionalized nanospears are guided magnetically to approach target adherent U87 glioblastoma cells, penetrating the cell membrane to enable intracellular delivery of the plasmid cargo. After 24 h, the target cell expresses green fluorescence indicating successful transfection. This nanospear-mediated transfection is readily scalable for the simultaneous manipulation of multiple cells using a rotating magnet. Cell viability >90% and transfection rates >80% were achieved, which exceed conventional nonviral intracellular methods. This approach is compatible with good manufacturing practices, circumventing barriers to the translation and clinical deployment of emerging cellular therapies.