In this study, we prepared polystyrene (PS) nanofibers as hepatocytes culture substrates by electrospinning method and subsequently coated with specific ligand (poly(N--vinylbenzyl-- β-D-galactopyranosyl-(14)-D-gluconamide)(PVLA) for hepatocytes attachment. Rat hepatocytes’ behavior on the PVLA-coated and non-coated PS nanofibrous matrices have been investigated. Electrospun PS fiber structures revealed randomly aligned fibers with average diameter of 500 nm. Fabricated PS nanofibers had no bonding points like cotton fibers. Analyses by ATR/FTIR and ESCA revealed that PVLA was successfully coated to the surfaces of PS nanofibers. More hepatocytes were attached on the surface of PS nanofibers coated with PVLA than that on noncoated PS nanofibers. PS nanofibrous matrix could incorporate many cells into the interior of the matrix probably due to the suitable pore size. Cell viabilities cultured on PVLA-coated PS nanofibrous mats were maintained for 2 weeks, while it was decreased rapidly on PVLA-coated PS dishes. High hepatic function especially albumin secretion was maintained for 2 weeks on nanofibrous mats but rapidly decreased on flat PS dishes. These results indicate that nanofibrous structure enabled spheroid-like culture results in providing cell-cell communication and subsequent long-term maintenance of specific cell function.