At present, the horse or human rabies immunoglobulin (RIG) used for postexposure prevention of human rabies (PEP) has high cost and limited availability. It is strongly encouraged to replace RIG with equivalent or more effective and safer products. Mouse and human monoclonal antibodies have been shown to protect rodents from lethal rabies virus (RABV) attacks. In this study, we reported a human-mouse chimeric monoclonal antibody, 12-2A12, which showed a strong neutralization potency and a wide breadth against multiple street viruses of RABV in vitro. The antibody binded the viral glycoprotein (G) with nanomolar affinity. The complex structure of 12-2A12 bound to RABV G revealed that the antibody recognizes an epitope that partially overlaps with the recognition region for the nicotinic acetylcholine receptor (nAChR). The antibody therefore would interfere with the nAChR/G interaction to block the viral receptor binding. In addition, comparison of our complex structure with the G structure in the acidic state reveals a clear steric clash, highlighting that the antibody would further prevent the conformational changes of the viral glycoprotein that are essential for membrane fusion. In light of these functional and structural data, we believe that 12-2A12 might be developed to be included in an antibody cocktail for potential use in human rabies PEP.© 2023 Wiley Periodicals LLC.