Piezoelectric poly(vinylidene fluoride-trifluoroethylene) has demonstrated an ability to promote osteogenesis, and biomaterials with a chirality-patterned topological surface could enhance cellular osteogenic differentiation. In this work, we created a chirality-patterned surface potential distribution of CoFe2O4/poly(vinylidene fluoride-trifluoroethylene (CFO/P(VDF-TrFE)) membranes to explore their osteogenic response under no change in surface chemical and topology, attempting to further strengthen the ability of the membranes to promote osteogenesis. The chirality-patterned surface potential distribution was established by microdomain contact polarization with the help of sinistral/dextral-patterned ITO interdigital microelectrodes. In the in vitro evaluations, the mesenchymal stem cells showed a positive response in osteogenic differentiation to CFO/P(VDF-TrFE) membranes with both sinistral- and dextral-patterned surface potential distributions, however, the dextral-patterned distribution gave a stronger response than the sinistral-patterned one. And the in vivo evaluation showed a response tend in new bone tissue formation similar to the in vitro evaluations. The stronger response in osteogenic differentiation and osteogenesis for the CFO/P(VDF-TrFE) membrane with the dextral-patterned distributions may be attributed to that the intense interaction of the cells with the electrophysiological microenvironment appears due to a correspondingly higher expression of integrin α5β1, which significantly up-regulates the Arp2/3 complex expression, a crucial factor for cytoskeleton reorganization, possibly increases cytoskeleton contractility, and strengthens the transduction of the osteogenesis-related signaling cascade. This work proves that the chirality-patterns in surface potential distributions could provide an osteogenic response similar to a chirality-patterned topological surface.