Nonwoven poly(epsilon-caprolactone) (PCL) nanofibers were prepared by electrospinning technology, and a novel natural bioactive material, soluble eggshell membrane protein (SEP), which is made from natural eggshell membrane (ESM), was then immobilized on the nanofibers after the surface modification. The SEP-immobilized fibrous mat was observed and characterized using scanning electron microscopy (SEM), contact angle measurement, Fourier transform infrared attenuated total reflection spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and tensile mechanical tests. Then the primary human dermal fibroblasts (HDFs) were cultured to evaluate the in vitro biocompatibility of SEP-grafted electrospun PCL nanofibers. The results confirmed the successful immobilization of SEP on the nanofibers and also indicated that the hydrophilicity of the PCL nanofibers has been greatly improved by the SEP grafting. The results of MTT testing, SEM, and laser scanning confocal microscope (LSCM) showed that SEP immobilization can obviously enhance the attachment, spreading, and proliferation of human dermal fibroblasts (HDFs) compared with the pristine material. The SEP-grafted PCL nanofibers can be expected to biomimic and regenerate the natural structure of eggshell membrane and to be a potential material for tissue engineering scaffold and guided tissue regeneration barrier membrane. (C) 2007 Wiley Periodicals, Inc.