An electrospinning method was used to fabricate silk fibroin (SF) nanofiber nonwovens for cell culture of normal human keratinocytes and fibroblasts. The electrospinning of regenerated SF was performed with formic acid as a spinning solvent. For insolubilization, as-spun SF nanofiber nonwovens were chemically treated with an aqueous methanol solution of 50%. Morphology and microstructure of as-spun and chemically treated SF nanofibers were investigated by scanning electron microscopy and mercury porosimetry. As-spun SF nanofibers exhibited a circular cross-section with a smooth surface. From the image analysis, they had an average diameter of 80 nm and their diameters ranged from 30 to 120 nm. During the chemical treatment for 60 min, porosity of nonwovens composed of SF nanofibers decreased from 76.1% up to 68.1%. To assay the cytocompatibility and cell behavior onto the electrospun SF nanofibers, cell attachment and spreading of normal human keratinocytes and fibroblasts seeded on the SF nanofibers and interaction between cells and SF nanofibers were studied. Cell morphology on SF nanofibers was examined by scanning electron microscopy. Our results indicate that the SF nanofibers may be a good candidate for the biomedical applications, such as wound dressing and scaffolds for tissue engineering.