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Programmable Cell Orientation Induced by Highly Ordered Gold Nanosphere Arrays Cellular orientation is critical for the formation and functionality of many native tissues, which plays a pivotal role in tissue engineering and regeneration. Here, an anisotropic surface nanostructure composed of highly ordered linear assemblies of gold nanospheres (AuNSs) on wrinkled polydimethylsiloxane substrates is fabricated to direct cell alignment. The AuNS linear assemblies provide nanotopographical cues that induce pronounced elongation and directional alignment of RSC96 Schwann cells along the AuNS chains, overriding the guidance effect of the underlying wrinkle topography. Immunofluorescence analysis further reveals that this guidance effect is associated with the directional organization of focal adhesions and tension-dependent mechanotransduction, involving myosin II-mediated cytoskeletal reorganization. This cell-guidance effect is also observed across multiple cell types, including L929 fibroblasts, HeLa cells, and bone marrow mesenchymal stem cells, demonstrating broad applicability across multiple cell types. Furthermore, the patterned AuNS assemblies could enable programmable control over cell alignment and migration. This surface nanostructure achieved by AuNS linear assemblies provides a potential strategy for manipulating cellular architecture and advancing the tissue engineering and regenerative medicine. https://pubs.acs.org/doi/10.1021/acsami.6c08528