The geometric properties of extracellular matrix (ECM) fibers play a crucial role in regulating cellular behaviors and functions. Although extensive research has examined the effects of fiber alignment, conflicting results have often arisen, leaving the precise mechanisms by which electrospun fiber alignment affects cellular behavior still unclear. This study investigates how the arrangement of polycaprolactone (PCL) electrospun fiber substrates affects cellular mechanosensing by modulating cell positioning. Larger cells, whose width on a coverslip exceeds 5 times the width of the aligned fiber gaps (≈8 µm in this study) and that span multiple aligned fibers, demonstrate enhanced spreading and mechanotransduction. Conversely, smaller cells, whose width is less than or equal to 2.5 times the width of the aligned fiber gaps and are confined within fiber interstices, exhibit limited mechanotransductive signaling. These findings are further supported by manipulating cell size and, more importantly, have led to the fabrication of semi-aligned fiber networks that enhance both cell spreading and mechanotransduction. This research emphasizes the importance of optimizing fiber architecture to improve cellular interactions, offering valuable insights for the design of biomimetic scaffolds in tissue regeneration.