Chemically and biologically modified substrates for single-cell patterning have been studied extensively, but physically modified structures for single-cell patterning still need further study. In this paper, physically modified silicon nanostructures were introduced to study their effect on SHSY5Y cells. Double-beam double exposure laser interference lithography combined with metal-assisted etching (MACE) was used to fabricate the physically modified silicon nanostructures. It was found that the cells on the gratings stretched and grew orderly along the grating with a small cell area and almost the same cell length compared with those on the Si wafer (control group). While on the grids, the cells were round with limited spreading, grew independently and had the smallest cell area and cell length. Moreover, the localization ratio of cells adhered onto the areas of nanopillars in the grid structures with different periods has been investigated. The results suggest that the physically modified grid silicon nanostructures can regulate the single-cell localization growth and the rational design of substrate structures can maximize the single-cell localization ratio. The findings provide guidance for the design of physically modified nanostructures and regulating single cell patterning, and a better understanding of single-cell localized growth.