Living cells receive biochemical and physical information from the surrounding microenvironment and respond to this information. Multiscale hierarchical substrates with micro- and nanogrooves have been shown to mimic the native extracellular matrix (ECM) better than conventional nanopatterned substrates; therefore, substrates with hierarchical topographical cues are considered suitable for investigating the role of physical factors in tissue functions. In this study, precisely controllable, multiscale hierarchical substrates that could mimic the micro- and nanotopography of complex ECMs were fabricated and used to culture various cell types, including fibroblasts, endothelial cells, osteoblasts, and human mesenchymal stem cells. These substrates had both microscale wrinkles and nanoscale patterns and enhanced the alignment and elongation of all the cells tested. In particular, the nanotopography on the microscale wrinkles promoted not only the adhesion, but also the functions of the cells. These findings suggest that the hierarchical multiscale substrates effectively regulated cellular structure and functions and that they can be used as a platform for tissue engineering and regenerative medicine.