The purpose of this study was to prepare and characterize a three-dimensional scaffold consisting of porous nonwoven silk fibroin net/nano-apatite composite. The silk fibroin net was mineralized with apatite by alternative soaking in calcium and phosphate solutions. The scaffold exhibited a porous microstructure with open porosity (70-78%), with an average pore size of about 163 +/- 40.0 microm. The swelling ratio and water uptake were 4.55% and 81.93%, respectively, indicating excellent hydrophilicity. The nano-sized hydroxyapatite crystals were needle like, with a length of approximately 100-300 nm and a diameter of approximately 20-60 nm. The results of in vitro cell culture study using rat osteoblast cells demonstrated that the nonwoven silk fibroin net/nano-apatite composite showed excellent cytocompatibility for the growth of osteoblasts and had the capability to improve the viability of osteoblasts.