Silk fibroin films are used in tissue engineering due to their biocompatibility, optical clarity, and slow biodegradability. However, the relatively smooth surface and low permeability of these systems may limit some applications; thus, here, a method was developed to generate nano-pores in methanol or ethanol-treated silk fibroin films. The first step was to induce the formation of nanoparticles (50-300 nm diam.) in silk fibroin solutions by autoclaving. After drying in air, the films formed were treated to induce silk β-sheet structures, which condense the bulk silk phase and nanoparticles and phase separation and enlarge the space of bulk silk phase and nanoparticles. These films were then extracted with water to allow the condensed nanoparticles to escape, leaving homogeneous nano-pores (50-300 nm) in the silk fibroin matrix. The introduction of nano-pores resulted in enhanced permeability and minimized loss of the mechanical properties of the nano-porous silk fibroin films (NSFs) when compared to the un-autoclaving-treated silk fibroin films. NSFs promoted cell (human fibroblasts) proliferation and oxygen/nutrition perfusion and significantly enhanced the complete skin-thickness wound healing in a rat model, suggesting the potential use in tissue regeneration or as wound dressing biomaterials for clinical applications.