Nanostructured neural interface coatings have significantly enhanced recording fidelity in both implantable and in vitro devices. As such, nanoporous gold (np‐Au) has shown promise as a multifunctional neural interface coating due, in part, to its ability to promote nanostructure‐mediated reduction in astrocytic surface coverage while not affecting neuronal coverage. The goal of this study is to provide insight into the mechanisms by which the np‐Au nanostructure drives the differential response of neurons versus astrocytes in an in vitro model. Utilizing microfabricated libraries that display varying feature sizes of np‐Au, it is demonstrated that np‐Au influences neural cell coverage through modulating focal adhesion formation in a feature size‐dependent manner. The results here show that surfaces with small (≈30 nm) features control astrocyte spreading through inhibition of focal adhesion formation, while surfaces with large (≈170 nm and greater) features control astrocyte spreading through other mechanotransduction mechanisms. This cellular response combined with lower electrical impedance of np‐Au electrodes significantly enhances the fidelity and stability of electrophysiological recordings from cortical neuron‐glia co‐cultures relative to smooth gold electrodes. Finally, by leveraging the effect of nanostructure on neuronal versus glial cell attachment, the use of laser‐based nanostructure modulation is demonstrated for selectively patterning neurons with micrometer spatial resolution.