An electrospun polysulfone (PSU) was prepared as a barrier membrane for guided bone regeneration. The membrane was in nanoscale to prevent fibrous tissue infiltration and highly porous to allow permeation of oxygen and nutrients. The morphology and attachment, viability and proliferation, and differentiation and mineralization of human bone marrow mesenchymal stem cells (HBMSCs) were determined. Cells adhered and spread well on the PSU membrane with characteristic polygonal, fusiform shapes and radial extensions. The live/dead staining revealed that the membrane had no negative influence on cell viability. The proliferation rates of HBMSCs on PSU membranes were lower in comparison with tissue-culture polystyrene plate after 3 days of culture. However, differentiation activity was particularly expressed at high levels when cells were cultured on PSU membranes. The results based on the data suggest that the PSU electrospun membrane promoted the osteogenic differentiation of HBMSCs, displayed desirable in vitro biocompatibility, and has good potential as a barrier membrane.