A chitosan/poly(L-lactic acid) (PLLA) multilayered membrane was prepared for the purpose of guided tissue regeneration (GTR). The membrane was composed of the outer layers of chitosan mesh and the middle layer of nanoporous PLLA membrane. The outer membranes of chitosan fibrous mesh present a highly biocompatible and rough surface for ease of cell adherence. The PLLA membrane is intended to provide mechanical strength to the membrane, thereby preventing epithelial invasion. The PLLA membrane was applied as a local drug delivery carrier for growth factors. Regular pores in PLLA membrane were generated through phase inversion of polymer solution. The membrane retained its integrity during the degradation process in vitro (for up to 8 weeks), which is a requirement for providing enough space in vivo in the GTR procedure. Osteoblasts firmly attached to broad cytoplasmic extensions along with the microfibers of the membrane. The phenotypic expression of cultured osteoblasts on the membrane was measured by a reverse transcriptase-polymerase chain reaction. New bone had formed beneath the chitosan/PLLA membrane. These results suggested that the chitosan/PLLA membranes properly functioned as biocompatible and mechanically stable barriers for GTR.