We modified hydroxyapatite (HA)-coated nonwoven polyethylene/polypropylene fabrics by coating with silk fibroin (SF) to improve them as a three-dimensional substrate for culturing human hepatocellular carcinoma-derived FLC-5 cells. After 25 days of culture, FLC-5 cells cultured on nonwoven fabrics coated with HA and HA plus SF (HA-SF) partially formed multicellular aggregates, whereas the cells cultured on tissue culture plates formed monolayers. FLC-5 cells cultured on nonwoven fabrics and tissue culture plates for 25 days were subjected to quantitative assay for cell number and albumin secretion. The lowest cell number and the largest amount of albumin were found for nonwoven fabrics coated with HA-SF. Normalizing albumin values to cell number data demonstrated that albumin secretory function per cell on 2 kinds of nonwoven fabrics was remarkably higher than that on tissue culture plates. Moreover, albumin secretory function per cell on nonwoven fabrics coated with HA-SF was twice as high as that on HA-coated nonwoven fabrics. These preliminary results suggest that modification of HA-coated nonwoven fabrics by SF coating induced functional improvement of FLC-5 cells.