The study of cell migration on biomaterials is of great significance in tissue engineering and regenerative medicine. In recent years, there has been increasing evidence that the physical properties of the extracellular matrix (ECM), such as surface topography, affect various cellular behaviors such as proliferation, adhesion, and migration. However, the biological mechanism of surface topography influencing cellular behavior is still unclear. In this study, we prepared polycaprolactone (PCL) fibrous materials with different surface microstructures by solvent casting, electrospinning, and self-induced crystallization. The corresponding topographical structure obtained is a two-dimensional (2D) flat surface, 2.5-dimensional (2.5D) fibers, and three-dimensional (3D) fibers with a multilevel microstructure. We then investigated the effects of the complex topographical structure on endothelial cell migration. Our study demonstrates that cells can sense the changes of micro- and nanomorphology on the surface of materials, adapt to the physical environment through biochemical reactions, and regulate actin polymerization and directional migration through Rac1 and Cdc42. The cells on the nanofibers are elongated spindles, and the positive feedback of cell adhesion and actin polymerization along the fiber direction makes the plasma membrane continue to protrude, promoting cell polarization and directional migration. This study might provide new insights into the biomaterial design, especially those used for artificial vascular grafts.