The biofunctionalization of materials creates interfaces on which proteins, cells, or tissues can fulfill native or desired tasks. Here we report how to control the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Collagen fibrils in these ultrathin (approximately 3 nm) matrices maintained their native structure as observed in vivo. This opens up the possibility to create programmable biofunctionalized matrices using collagen-binding proteins or proteins fused with collagen-binding domains. Applied to eukaryotic cells, these nanostructured matrices can direct cellular processes such as adhesion, orientation and migration.