Topographical properties, such as pattern and diameter, of biomaterials play important roles in influencing cell activities and manipulating the related immune response during wound healing. We prepared aligned electrospinning membranes with different fiber diameters, including 319 ± 100 nm (A300), 588 ± 132 nm (A600), and 1048 ± 130 nm (A1000), by adjusting the distance from the tip to the collector, the injection rate, and the concentration of the solution. The A300 membranes significantly improved cell proliferation and spreading and facilitated wound healing (epithelization and vascularization) with the regeneration of immature hair follicles compared to the other membranes. Transcriptomics revealed the underlying molecular mechanism that A300 could promote immune-related processes towards a pro-healing direction, significantly promoting keratinocyte migration and skin wound healing. All the results indicated that wound healing requires the active participation of the immune process, and that A300 was a potential candidate for guided skin regeneration applications.