The barrier membranes maintain a secluded space to prevent the ingrowth of connective tissue and direct the growth of new bone into a desired site; however, they do not stimulate or induce bone regeneration. To enhance the bone bioactivities of membranes, we developed chitosan electret membranes with bioelectricity by grid-controlled constant voltage corona charging. The electret membranes charged with heat treatment (HT electret membranes) exhibited superior electret charge storage stability than the ones charged without heat treatment (RT electret membranes). Human bone marrow stromal cells (hBMSCs) demonstrated better growth on HT electrets membrane. Moreover, hBMSCs osteoblastic differentiation was enhanced on HT electret membranes, as evidenced by osteocalcin and osteopontin expression as assessed by immunocytochemistry, quantitative RT-PCR and western blot analysis. The rabbit calvarial defect model demonstrated that HT electret membranes induced a significantly enhanced bone regeneration compared with RT electret membranes. New bone formation was found at both the periphery and in the center of the defects four weeks after implantation. These results indicated that the chitosan electret membrane has osteogenic potential and could be applied as a novel barrier membrane.